Molecules with extended half-lives, compositions and uses thereof

ABSTRACT

The present invention provides molecules, including IgGs, non-IgG immunoglobulin, proteins and non-protein agents, that have increased in vivo half-lives due to the presence of an IgG constant domain, or a portion thereof that binds the FcRn, having one or more amino acid modifications that increase the affinity of the constant domain or fragment for FcRn. Such proteins and molecules with increased half-lives have the advantage that smaller amounts and or less frequent dosing is required in the therapeutic, prophylactic or diagnostic use of such molecules.

[0001] This application claims the benefit of U.S. provisionalapplication Serial Nos. 60/254,884, filed Dec. 12, 2000, and 60/289,760,filed May 9, 2001, both of which are incorporated by reference herein intheir entireties. This invention was made, in part, with United StatesGovernment support under award number A139167 from the NationalInstitute of Health. The United States Government may have certainrights in the invention.

1. INTRODUCTION

[0002] The present invention relates to molecules whose in vivohalf-lives are increased by modification of an IgG constant domain, orFcRn (Fc Receptor-neonate) binding domain thereof. Specifically, thesemolecules have amino acid modifications that increase the affinity ofthe constant domain or fragment thereof for the FcRn. Increasing thehalf-life of therapeutic and diagnostic IgGs and other bioactivemolecules using methods of the invention has many benefits includingreducing the amount and/or frequency of dosing of these molecules, forexample, in vaccines, passive immunotherapy and other therapeutic andprophylactic methods. The invention further relates to fusion proteinscontaining all or a portion (a FcRn binding portion) of an IgG constantdomain having one or more of these amino acid modifications and anon-IgG protein or non-protein molecule conjugated to such a modifiedIgG constant domain, where the presence of the modified IgG constantdomain increases the in vivo half-life of the non-IgG protein ormolecule.

2. BACKGROUND OF THE INVENTION

[0003] The use of immunoglobulins as therapeutic agents has increaseddramatically in recent years and have expanded to different areas ofmedical treatments. Such uses include treatment of agammaglobulinemiaand hypogammaglobulinemia, as immunosuppressive agents for treatingautoimmune diseases and graft-vs.-host (GVH) diseases, the treatment oflymphoid malignancies, and passive immunotherapies for the treatment ofvarious systemic and infectious diseases. Also, immunoglobulins areuseful as in vivo diagnostic tools, for example, in diagnostic imagingprocedures.

[0004] One critical issue in these therapies is the persistence ofimmunoglobulins in the circulation. The rate of immunoglobulin clearancedirectly affects the amount and frequency of dosage of theimmunoglobulin. Increased dosage and frequency of dosage may causeadverse effects in the patient and also increase medical costs.

[0005] IgG is the most prevalent immunoglobulin class in humans andother mammals and is utilized in various types of immunotherapies anddiagnostic procedures. The mechanism of IgG catabolism in thecirculation has been elucidated through studies related to the transferof passive immunity from mother to fetus/neonate through the placenta oryolk sac or through colostrum (maternofetal transfer of IgG viatranscytosis) in rodents (Brambell, Lancet, ii: 1087-1093, 1966;Rodewald, J. Cell Biol., 71:666-670, 1976; Morris et al., In: AntigenAbsorption by the Gut, pp. 3-22, 1978, University Park Press, Baltimore;Jones et al., J. Clin. Invest., 51:2916-2927, 1972).

[0006] The involvement of certain receptors in the maternofetaltransmission of maternal IgGs was first suggested by Brambell's group intheir study on the intestinal absorption of maternal antibodies fromingested milk in newborn rats (Halliday, Proc. R. Soc. B., 143:408-413,1955; Halliday, Proc. R. Soc. B., 144:427-430, 1955; Halliday, Proc. R.Soc. B., 148:92-103, 1957; Morris, Proc. R. Soc. B., 148:84-91, 1957;Brambell et al., Proc. R. Soc. B., 149:1-11, 1958; Morris, Proc. R. Soc.B., 160:276-292, 1964). Brambell et al. suggested, based on theobservation that heterologous IgGs interfered with the transmission of aspecific antibody, that IgG molecules from various species might havesufficiently similar structures or sequences that bind to commonreceptors (Brambell et al., Proc. R. Soc. B., 149:1-11, 1958).

[0007] A high-affinity Fc receptor, FcRn, has been implicated in thistransfer mechanism. The FcRn receptor has been isolated from duodenalepithelial brush borders of suckling rats (Rodewald et al., J. CellBiol., 99:154s-164s, 1984; Simister et al., Eur. J. Immunol.,15:733-738, 1985) and the corresponding gene has been cloned (Simisteret al., Nature, 337:184, 1989 and Cold Spring Harbor Symp. Quant. Biol.,LIV, 571-580, 1989). The later clonings of FcRn-encoding genes from mice(Ahouse et al., J. Immunol., 151:6076-6088, 1993) and humans (Story etal., J. Exp. Med., 180:2377-2381, 1994) demonstrate high homology ofthese sequences to the rat FcRn, suggesting a similar mechanism ofmaternofetal transmission of IgGs involving FcRn in these species.

[0008] Meanwhile, a mechanism for IgG catabolism was also proposed byBrambell's group (Brambell et al., Nature, 203:1352-1355, 1964;Brambell, Lancet, ii: 1087-1093, 1966). They proposed that a proportionof IgG molecules in the circulation are bound by certain cellularreceptors (i. e., FcRn), which are saturable, whereby the IgGs areprotected from degradation and eventually recycled into the circulation;on the other hand, IgGs which are not bound by the receptors aredegraded. The proposed mechanism was consistent with the IgG catabolismobserved in hypergammaglobulinemic or hypogammaglobulinemic patients.Furthermore, based on his studies as well as others (see, e.g.,Spiegelberg et al., J. Exp. Med., 121:323-338, 1965; Edelman et al.,Proc. Natl. Acad. Sci. USA, 63:78-85, 1969), Brambell also suggestedthat the mechanisms involved in maternofetal transfer of IgG andcatabolism of IgG may be either the same or, at least, very closelyrelated (Brambell, Lancet, ii:1087-1093, 1966). Indeed, it was laterreported that a mutation in the Fc-hinge fragment caused concomitantchanges in catabolism, maternofetal transfer, neonatal transcytosis,and, particularly, binding to FcRn (Ghetie et al., Immunology Today,18(12):592-598, 1997).

[0009] These observations suggested that portions of the IgG constantdomain control IgG metabolism, including the rate of IgG degradation inthe serum through interactions with FcRn. Indeed, increased bindingaffinity for FcRn increased the serum half-life of the molecule (Kim etal., Eur. J. Immunol., 24:2429-2434, 1994; Popov et al., Mol. Immunol.,33:493-502, 1996; Ghetie et al., Eur. J. Immunol., 26:690-696, 1996;Junghans et al., Proc. Natl. Acad. Sci. USA, 93:5512-5516, 1996; Israelet al., Immunol., 89:573-578, 1996).

[0010] Various site-specific mutagenesis experiments in the Fc region ofmouse IgGs have led to identification of certain critical amino acidresidues involved in the interaction between IgG and FcRn (Kim et al.,Eur. J. Immunol., 24:2429-2434, 1994; Medesan et al., Eur. J. Immunol.,26:2533, 1996; Medesan et al., J. Immunol., 158:2211-2217, 1997). Thesestudies and sequence comparison studies found that isoleucine atposition 253, histidine at position 310, and histidine at position 435(according to Kabat numbering, Kabat et al., In: Sequences of Proteinsof Immunological Interest, US Department of Health and Human Services,1991, which is hereby incorporated by reference in its entirety), arehighly conserved in human and rodent IgGs, suggesting their importancein IgG-FcRn binding.

[0011] Additionally, various publications describe methods for obtainingphysiologically active molecules whose half-lives are modified either byintroducing an FcRn-binding polypeptide into the molecules (WO 97/43316;U.S. Pat. No. 5,869,046; U.S. Pat. No. 5,747,035; WO 96/32478; WO91/14438) or by fusing the molecules with antibodies whose FcRn-bindingaffinities are preserved but affinities for other Fc receptors have beengreatly reduced (WO 99/43713) or fusing with FcRn binding domains ofantibodies (WO 00/09560; U.S. Pat. No. 4,703,039). However, none ofthese publications disclose specific mutants in the IgG constant domainthat affect half-life.

[0012] Prior studies have demonstrated that certain constant domainmutations actually reduce binding to FcRn and, thereby, reduce the IgGin vivo half-life. PCT publication WO 93/22332 (by Ward et al.)discloses various recombinant mouse IgGs whose in vivo half-lives arereduced by mutations between about residue 253 and about residue 434.Particularly, substitutions of isoleucine at position 253; histidine atposition 310; glutamine at position 311; His at position 433; andasparagine at position 434 were found to reduce IgG half-life.

[0013] Modulation of IgG molecules by amino acid substitution, addition,or deletion to increase or reduce affinity for FcR-n is also disclosedin WO 98/23289; however, the publication does not list any specificmutants that exhibit either longer or shorter in vivo half-lives.

[0014] In fact, only one mutant of mouse IgGI that actually exhibitedincreased half-life, the triple mutation Thr252 to Ala, Thr254 to Ser,and Thr256 to Phe, has been identified (WO 97/34631).

[0015] In view of the pharmaceutical importance of increasing the invivo half-lives of immunoglobulins and other bioactive molecules, thereis a need to develop modified IgGs and FcRn-binding fragments thereof,(particularly modified human IgGs) that confer increased in vivohalf-life on immunoglobulins and other bioactive molecules.

3. SUMMARY OF THE INVENTION

[0016] The present invention is based upon the inventors' identificationof several mutations in the constant domain of a human IgG molecule thatincrease the affinity of the IgG molecule for the FcRn. In particular,the present inventors have screened libraries of human IgG1 constantdomains with random amino acid mutations introduced into particularregions of the constant domain for increased affinity for FcRn. Suchrandom mutations were made in the regions of residues 251-256, 285-290,and 308-314, all of which are in CH2 domain, and 385-389 and 428-436,which are in CH3 domain, of human IgG1 hinge-Fc regions (residues asdepicted in FIG. 2 (SEQ ID NO:83 or analogous residues in hinge-Fcregions of other IgG molecules as determined by sequence alignment). Asused herein, all residues of the IgG constant domain are numberedaccording to Kabat et al. (Sequences of Proteins of ImmunologicalInterest, U.S. Department of Health and Human Services, 1991, which isincorporated by reference herein in its entirety) and as presented inFIG. 2 (SEQ ID NO:83), and include corresponding residues in other IgGconstant domains as determined by sequence alignment. The in vivohalf-life, or persistence in serum or other tissues of a subject, ofantibodies, and other therapeutic agents and other bioactive moleculesis an important clinical parameter which determines the amount andfrequency of antibody (or any other pharmaceutical molecule)administration. Accordingly, such molecules, including antibodies, withincreased half-life are of significant pharmaceutical importance.

[0017] Thus, the present invention relates to a modified molecule(preferably a protein, but may be a non-protein agent) that has anincreased in vivo half-life by virtue of the presence of a modified IgGconstant domain, or FcRn-binding portion thereof (preferably the Fc orhinge-Fc domain) (preferably from a human IgG) wherein the IgG constantdomain, or fragment thereof, is modified (e.g., by amino acidsubstitution, deletion or insertion) to increase the affinity for theFcRn. In a particular embodiment, the present invention relates tomodified IgGs, whose in vivo half-lives are extended by the modificationof amino acid residues identified to be involved in the interaction ofthe hinge-Fc domain with the FcRn receptor. Preferably, the constantdomain or fragment thereof has higher affinity for FcRn at pH 6.0 thanat pH 7.4. Such modifications may also alter (i.e., increase ordecrease) the bioavailability (e.g., transport to mucosal surfaces, orother target tissues) of the molecules. The invention also relates toother types of immunoglobulins or fragments thereof (i.e., non-IgGimmunoglobulins), non-immunoglobulin proteins and non-protein agentsthat are fused or conjugated to, or engineered to contain, an IgGconstant domain, or FcRn-binding fragment thereof, having one or moresuch amino acid modifications.

[0018] In preferred embodiments, the present invention providesmolecules, particularly, immunoglobulins whose in vivo half-lives areextended by the presence of an IgG constant domain, or FcRn bindingfragment thereof (preferably, Fc or hinge-Fc domain), that hasmodifications of one or more of amino acid residues 251-256, 285-290,308-314, 385-389, and 428-436 that increase the affinity of the constantdomains or fragments thereof for FcRn. In certain embodiments, thesemodifications preferably exclude residues 252, 254, and 256, inparticular when the IgG constant domain or fragment thereof, is murine.In particular embodiments, the modification is at one or moresurface-exposed residues, and the modification is a substitution with aresidue of similar charge, polarity or hydrophobicity to the residuebeing substituted. In preferred embodiments, the modified IgG constantdomain, or fragment thereof, binds with higher affinity to FcRn at pH6.0 than at pH 7.4. In a preferred embodiment, the constant domain, orfragment hereof, is modified by substitution of one or more of aminoacid residues 251-256, 285-290, 308-314, 385-389, and 428-436 thatincrease the affinity of the constant domain or FcRn-binding fragmentsthereof for FcRn. In certain embodiments, substitutions of residue 252with leucine, residue 254 with serine, and/or residue 256 withphenylalanine are excluded, particularly when the constant domain orfragment thereof is derived from a mouse IgG.

[0019] In specific embodiments, the invention provides immunoglobulinsor other bioactive molecules that contain an IgG1 constant domain, orFcRn-binding fragment thereof (preferably Fc or hinge-Fc domain)(preferably human), having amino acid modifications at one or more ofposition 308, 309, 311, 312, and 314, more specifically, havingsubstitutions at one or more of positions 308, 309, 311, 312 and 314with threonine, proline, serine, aspartic acid and leucine respectively.In another embodiment, residues at one or more of positions 308, 309,and 311 are substituted with isoleucine, proline, and glutamic acid,respectively. In yet another embodiment, residues at one or more ofpositions 308, 309, 311, 312, and 314, are substituted with threonine,proline, serine, aspartic acid, and leucine, respectively. The inventionfurther relates to combinations of these amino acid substitutions.

[0020] Furthermore, the invention provides immunoglobulins or otherbioactive molecules that contain an IgG1 constant domain, orFcRn-binding fragment thereof (preferably, Fc or hinge-Fc domain)(preferably human), having amino acid modifications at one or more ofpositions 251, 252, 254, 255, and 256, more specifically, havingsubstitutions at one or more of these positions. In specificembodiments, residue 251 is substituted with leucine or arginine,residue 252 is substituted with tyrosine, phenylalanine, serine,tryptophan or threonine, residue 254 is substituted with threonine orserine, residue 255 is substituted with leucine, glycine, isoleucine orarginine, and/or residue 256 is substituted with serine, arginine,glutamine, glutamic acid, aspartic acid, alanine, asparagine orthreonine. In a more specific embodiment, residue 251 is substitutedwith leucine, residue 252 is substituted with tyrosine, residue 254 issubstituted with threonine or serine, and/or residue 255 is substitutedwith arginine. In yet another specific embodiment, residue 252 issubstituted with phenylalanine and/or residue 256 is substituted withaspartic acid. In a preferred embodiment, residue 251 is substitutedwith leucine, residue 252 is substituted with tyrosine, residue 254 issubstituted with threonine or serine, and/or residue 255 is substitutedwith arginine. The invention further relates to any combination of thesesubstitutions.

[0021] Furthermore, the invention provides immunoglobulins or otherbioactive molecules that contain an IgG1 constant domain, orFcRn-binding fragment thereof (preferably, Fc or hinge-Fc domain)(preferably human), having amino acid modifications at one or more ofpositions 428, 433, 434, and 436, more specifically, havingsubstitutions at one or more of these positions. In specificembodiments, residue 428 is substituted with methionine, threonine,leucine, phenylalanine, or serine, residue 433 is substituted withlysine, arginine, serine, isoleucine, proline, glutamine, or histidine,residue 434 is substituted with phenylalanine, tyrosine, or histidine,and/or residue 436 is substituted with histidine, asparagine, arginine,threonine, lysine, methionine, or threonine. In a more specificembodiment, residues at one or more positions 433, 434, and 436 aresubstituted with lysine, phenylalanine, and histidine, respectively. Ina preferred embodiment, residue 428 is substituted with methionineand/or residue 434 is substituted with tyrosine.

[0022] Furthermore, the invention provides immunoglobulins or otherbioactive molecules that contain an IgG1 constant domain, orFcRn-binding fragment thereof (preferably, Fc or hinge-Fc domain)(preferably human), having amino acid modifications at one or morepositions 385, 386, 387, and 389, more specifically, havingsubstitutions at one or more of these positions. In specificembodiments, residue 385 is substituted with arginine, aspartic acid,serine, threonine, histidine, lysine, or alanine, residue 386 issubstituted with threonine, proline, aspartic acid, serine, lysine,arginine, isoleucine, or methionine, residue 387 is substituted witharginine, histidine, serine, threonine, alanine, or proline and/orresidue 389 is substituted with proline or serine. In more specificembodiments, residues at one or more positions 385, 386, 387, and 389are substituted with arginine, threonine, arginine, and proline,respectively. In yet another specific embodiment, residues at one ormore positions 385, 386, and 389 are substituted with aspartic acid,proline, and serine, respectively.

[0023] Molecules of the invention include any combination of theabove-described substitutions at one or more of residues 251, 252, 254,255, 256, 308, 309, 311, 312, 385, 386, 387, 389, 428, 433, 434, and/or436. In a preferred embodiment, the molecule of the invention contains aFc region, or FcRn-binding domain thereof, having one or more of thefollowing substitutions: leucine at residue 251, tyrosine at residue252, threonine or serine at residue 254, arginine at residue 255,threonine at residue 308, proline at residue 309, serine at residue 311,aspartic acid at residue 312, leucine at residue 314, arginine atresidue 385, threonine at residue 386, arginine at residue 387, prolineat residue 389, methionine at residue 428, and/or tyrosine at residue434.

[0024] Included within the invention are pharmaceutical compositions andmethods of prophylaxis and therapy using modified immunoglobulins,proteins and other bioactive molecules of the invention having extendedhalf-lives. Also included are methods of diagnosis using modifiedimmunoglobulins, proteins and other bioactive molecules of the inventionhaving extended half-lives. In a specific embodiment, the inventionprovides an anti-respiratory syncytial virus (RSV) antibody useful totreat or prevent RSV infection, such as SYNAGIS® (see U.S. Pat. No.5,824,307 and Johnson et al., J. Infectious Disease 176:1215-1224, 1997,both of which are incorporated by reference in their entireties), andother anti-RSV antibodies, including variants of SYNAGIS® (see U.S.patent application Ser. No., 09/724,396, filed Nov. 28, 2000, U.S.patent application Ser. No. 09/724,531, filed Nov. 28, 2000, U.S. patentapplication Ser. No. ______, filed Nov. 28, 2001 (attorney docket no.10271-047), and U.S. patent application Ser. No. ______, filed Nov. 28,2001 (attorney docket no. 10271-048), all entitled “Methods ofAdministering/Dosing Anti-RSV Antibodies for Prophylaxis and Treatment,”all by Young et al., all of which are incorporated by reference hereinin their entireties, particularly the sequences of heavy and light chainvariable domains and CDRs of anti-RSV antibodies disclosed therein),which has one or more amino acid modifications in the constant domainthat increase the affinity of the antibody for FcRn and that has anincreased in vivo half-life (see also, Section 5.1 infra).

3.1 DEFINITIONS

[0025] The term “IgG Fc region” as used herein refers the portion of anIgG molecule that correlates to a crystallizable fragment obtained bypapain digestion of an IgG molecule. The Fc region consists of theC-terminal half of the two heavy chains of an IgG molecule that arelinked by disulfide bonds. It has no antigen binding activity butcontains the carbohydrate moiety and the binding sites for complementand Fc receptors, including the FcRn receptor (see below). The Fcfragment contains the entire second constant domain CH2 (residues231-340 of human IgG1, according to the Kabat numbering system) (e.g.,SEQ ID NO:80) and the third constant domain CH3 (residues 341-447) (e.g,SEQ ID NO:81).

[0026] The term “IgG hinge-Fc region” or “hinge-Fc fragment” as usedherein refers to a region of an IgG molecule consisting of the Fc region(residues 231-447) and a hinge region (residues 216-230; e.g., SEQ IDNO:82) extending from the N-terminus of the Fc region. An example of theamino acid sequence of the human IgG1 hinge-Fc region is SEQ ID NO:83.

[0027] The term “constant domain” refers to the portion of animmunoglobulin molecule having a more conserved amino acid sequencerelative to the other portion of the immunoglobulin, the variabledomain, which contains the antigen binding site. The constant domaincontains the CH1, CH2 and CH3 domains of the heavy chain and the CHLdomain of the light chain.

[0028] The term “FcRn receptor” or “FcRn” as used herein refers to an Fcreceptor (“n” indicates neonatal) which is known to be involved intransfer of maternal IgGs to a fetus through the human or primateplacenta, or yolk sac (rabbits) and to a neonate from the colostrumthrough the small intestine. It is also known that FcRn is involved inthe maintenance of constant serum IgG levels by binding the IgGmolecules and recycling them into the serum. The binding of FcRn to IgGmolecules is strictly pH-dependent with optimum binding at pH 6.0. FcRncomprises a heterodimer of two polypeptides, whose molecular weights areapproximately 50 kD and 15 kD, respectively. The extracellular domainsof the 50 kD polypeptide are related to major histocompatibility complex(MHC) class I α-chains and the 15 kD polypeptide was shown to be thenon-polymorphic β₂-microglobulin (β₂-m). In addition to placenta andneonatal intestine, FcRn is also expressed in various tissues acrossspecies as well as various types of endothelial cell lines. It is alsoexpressed in human adult vascular endothelium, muscle vasculature andhepatic sinusoids and it is suggested that the endothelial cells may bemost responsible for the maintenance of serum IgG levels in humans andmice. The amino acid sequences of human FcRn and murine FcRn areindicated by SEQ ID NO:84 and SEQ ID NO:85, respectively. Homologs ofthese sequences having FcRn activity are also included.

[0029] The term “in vivo half-life” as used herein refers to abiological half-life of a particular type of IgG molecule or itsfragments containing FcRn-binding sites in the circulation of a givenanimal and is represented by a time required for half the quantityadministered in the animal to be cleared from the circulation and/orother tissues in the animal. When a clearance curve of a given IgG isconstructed as a function of time, the curve is usually biphasic with arapid α-phase which represents an equilibration of the injected IgGmolecules between the intra- and extra-vascular space and which is, inpart, determined by the size of molecules, and a longer β-phase whichrepresents the catabolism of the IgG molecules in the intravascularspace. The term “in vivo half-life” practically corresponds to the halflife of the IgG molecules in the β-phase.

[0030] An “isolated” or “purified” antibody or fusion protein issubstantially free of cellular material or other contaminating proteinsfrom the cell or tissue source from which the protein is derived, orsubstantially free of chemical precursors or other chemicals whenchemically synthesized. The language “substantially free of cellularmaterial” includes preparations of an antibody or a fusion protein inwhich the antibody or the fusion protein is separated from cellularcomponents of the cells from which it is isolated or recombinantlyproduced. Thus, an antibody or a fusion protein that is substantiallyfree of cellular material includes preparations of antibody or fusionprotein having less than about 30%, 20%, 10%, or 5% (by dry weight) ofcontaminating protein. When the antibody or the fusion protein isrecombinantly produced, it is also preferably substantially free ofculture medium, i.e., culture medium represents less than about 20%,10%, or 5% of the volume of the protein preparation. When the antibodyor the fusion protein is produced by chemical synthesis, it ispreferably substantially free of chemical precursors or other chemicals,i.e., it is separated from chemical precursors or other chemicals whichare involved in the synthesis of the protein. Accordingly suchpreparations of the antibody or the fusion protein have less than about30%, 20%, 10%, 5% (by dry weight) of chemical precursors or compoundsother than the antibody or antibody fragment of interest. In a preferredembodiment of the present invention, antibodies are isolated orpurified. In another preferred embodiment of the invention, fusionproteins are isolated or purified.

[0031] An “isolated” nucleic acid molecule is one which is separatedfrom other nucleic acid molecules which are present in the naturalsource of the nucleic acid molecule. Moreover, an “isolated” nucleicacid molecule, such as a cDNA molecule, can be substantially free ofother cellular material, or culture medium when produced by recombinanttechniques, or substantially free of chemical precursors or otherchemicals when chemically synthesized. An “isolated” nucleic acidmolecule does not include cDNA molecules within a cDNA library. In apreferred embodiment of the invention, nucleic acid molecules encodingantibodies are isolated or purified. In another preferred embodiment ofthe invention, nucleic acid molecules encoding fusion proteins areisolated or purified.

[0032] The term “host cell” as used herein refers to the particularsubject cell transfected with a nucleic acid molecule or infected withphagemid or bacteriophage and the progeny or potential progeny of such acell. Progeny of such a cell may not be identical to the parent celltransfected with the nucleic acid molecule due to mutations orenvironmental influences that may occur in succeeding generations orintegration of the nucleic acid molecule into the host cell genome.

[0033] The names of amino acids referred to herein are abbreviatedeither with three-letter or one-letter symbols.

[0034] To determine the percent identity of two amino acid sequences orof two nucleic acid sequences, the sequences are aligned for optimalcomparison purposes (e.g., gaps can be introduced in the sequence of afirst amino acid or nucleic acid sequence for optimal alignment with asecond amino acid or nucleic acid sequence). The amino acid residues ornucleotides at corresponding amino acid positions or nucleotidepositions are then compared. When a position in the first sequence isoccupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences (i.e., % identity=number of identical overlappingpositions/total number of positions×100%). In one embodiment, the twosequences are the same length.

[0035] The determination of percent identity between two sequences canalso be accomplished using a mathematical algorithm. A preferred,non-limiting example of a mathematical algorithm utilized for thecomparison of two sequences is the algorithm of Karlin and Altschul,1990, Proc. Natl. Acad. Sci. U.S.A. 87:2264-2268, modified as in Karlinand Altschul, 1993, Proc. Natl. Acad. Sci. U.S.A. 90:5873-5877. Such analgorithm is incorporated into the NBLAST and XBLAST programs ofAltschul et al., 1990, J. Mol. Biol. 215:403. BLAST nucleotide searchescan be performed with the NBLAST nucleotide program parameters set,e.g., for score=100, wordlength=12 to obtain nucleotide sequenceshomologous to a nucleic acid molecules of the present invention. BLASTprotein searches can be performed with the XBLAST program parametersset, e.g., to score-50, wordlength=3 to obtain amino acid sequenceshomologous to a protein molecule of the present invention. To obtaingapped alignments for comparison purposes, Gapped BLAST can be utilizedas described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402.Alternatively, PSI-BLAST can be used to perform an iterated search whichdetects distant relationships between molecules (Id.). When utilizingBLAST, Gapped BLAST, and PSI-Blast programs, the default parameters ofthe respective programs (e.g., of XBLAST and NBLAST) can be used (see,e.g., http://www.ncbi.nlm.nih.gov). Another preferred, non-limitingexample of a mathematical algorithm utilized for the comparison ofsequences is the algorithm of Myers and Miller, 1988, CABIOS 4:11-17.Such an algorithm is incorporated in the ALIGN program (version 2.0)which is part of the GCG sequence alignment software package. Whenutilizing the ALIGN program for comparing amino acid sequences, a PAM120weight residue table, a gap length penalty of 12, and a gap penalty of 4can be used.

[0036] The percent identity between two sequences can be determinedusing techniques similar to those described above, with or withoutallowing gaps. In calculating percent identity, typically only exactmatches are counted.

4. DESCRIPTION OF THE FIGURES

[0037]FIG. 1 shows the structure of the IgG hinge-Fc region indicatingthe locations of the residues identified to be involved in theinteraction with the FcRn receptor (Ghetie et al., Immunology Today,18(12):592-598, 1997).

[0038]FIG. 2 shows the amino acid sequence of the human IgG1 hinge-Fcregion (SEQ ID NO:83) containing a hinge region (SEQ ID NO:82), CH2domain (SEQ ID NO:80), and CH3 domain (SEQ ID NO:81).

[0039] FIGS. 3(A and B) show the amino acid sequences of (A) human FcRn(SEQ ID NO:84) and (B) mouse FcRn (SEQ ID NO:85), respectively.

[0040]FIG. 4 shows the amino acid sequence of the human IgG1 hinge-Fcregion (SEQ ID NO:83), in which wild-type residues which are mutated byamino acid substitutions are indicated in underlined bold-face.

[0041]FIG. 5 shows a schematic diagram of panning process for thephage-displayed modified hinge-Fc library.

[0042]FIG. 6 shows a summary of the occurrence of selected mutantresidues at the variant positions in the libraries screened.

[0043] FIGS. 7(A-D). (A) shows the binding of murine FcRn to immobilizedIgG1 having M252Y/S254T/T256E substitutions. Murine FcRn was injected at10 different concentrations ranging from 1 nM to 556 nM over a surfaceon which 4000 resonance units (RU) of IgG1 had been coupled. Afterequilibrium was reached, residual bound protein was eluted with a pulseof PBS, pH 7.4. (B) shows the binding of human FcRn to immobilizedIgG1/M252Y/S254T/T256E. Murine FcRn was injected at 8 differentconcentrations ranging from 71 nM to 2.86 μM over a surface on which1000 RU of IgG1 had been coupled. After equilibrium was reached,residual bound protein was eluted with a pulse of PBS, pH 7.4. (C) and(D) show scatchard analyses of the data in (A) and (B), respectively,after correction for nonspecific binding. R_(eq) is the correctedequilibrium response at a given concentration C. The plots are linearwith correlation coefficients of 0.97 and 0.998, respectively. Theapparent K_(d) are 24 nM and 225 nM, respectively.

[0044] FIGS. 8(A-H). (A)-(D) show the results from BIAcore analysis ofthe binding of murine FcRn at pH 6.0 and pH 7.4 to (A) wild type humanIgG1, (B) M252Y/S254T/T256E, (C) H433K/N434F/Y436H, and (D)G385D/G386P/N389S, respectively, after correction for nonspecificbinding. Murine FcRn was injected at a concentration of 1.1 μm over asurface on which 1000 RU of wild type IgG1, 1000 RU ofM252Y/S254T/T256E, 955 RU of H433K/N434F/Y436H, and 939 RU ofG385D/Q386P/N389S had been coupled. (E)-(H) show the results fromBIAcore analysis of the binding of human FcRn at pH 6.0 and pH 7.4 to(E) wild type human IgG1, (F) M252Y/S254T/T256E, (G) H433K/N434F/Y436H,and (H) G385D/Q386P/N389S, respectively, after correction fornonspecific binding. Human FcRn was injected at a concentration of 1.4μm over a surface on which 1000 RU of wild type IgG1, 1000 RU ofM252Y/S254T/T256E, 955 RU of H433K/N434F/Y436H, and 939 RU ofG385D/Q386P/N389S had been coupled.

[0045]FIG. 9 shows the space-filling model of the surface of the Fcfragment of a human IgG1 based upon the human IgG1 structure ofDeisenhofer, 1981, Biochemistry 20:2361-2370. Residues are color-codedaccording to the gain of free energy of stabilization of the Fc-FcRncomplex: red, substitutions at these positions were found to increaseaffinity b a factor of at least 2.5 times in the Fc/human FcRninteraction and of at least 5 time in the Fc/mouse FcRn interaction;blue, substitutions at those positions were found to increase affinityby a factor of less than 2 times in both the Fc-human FcRn and Fc-mouseFcRn interaction. The figure was drawn using Swiss pdb viewer (Guex andPeitsch, 1997, Electrophoresis 18:2714-2723).

[0046]FIG. 10 shows the changes in serum concentration ([Mab] ng/ml)over time (in days) of antibody having a wild type constant domain(SYNAGIS®) (open squares), or constant domains with the followingmutations: M252Y/S254T/T256E (open circles), G385D/Q386P/N389S (solidsquares), and H433K/N434F/Y436H (solid circles). Antibody concentrationwas determined using anti-human IgG ELISA.

5. DETAILED DESCRIPTION OF THE INVENTION

[0047] The present invention relates to molecules, particularlyproteins, more particularly immunoglobulins, that have an increased invivo half-life and comprise an IgG constant domain, or fragment thereofthat binds to an FcRn (preferably a Fc or hinge-Fc domain), thatcontains one or more amino acid modifications relative to a wild typeIgG constant domain which modifications increase the affinity of the IgGconstant domain, or fragment thereof, for the FcRn. In a preferredembodiment, the invention particularly relates to the modification ofhuman or humanized IgGs and other bioactive molecules containingFcRn-binding portions of human IgGs, which have particular use in humantherapy, prophylaxis and diagnosis.

5.1 MOLECULES WITH INCREASED IN VIVO HALF-LIVES

[0048] The present invention is based upon identification of amino acidmodifications in particular portions of the IgG constant domain thatinteract with the FcRn, which modifications increase the affinity of theIgG, or fragment thereof, for the FcRn. Accordingly, the inventionrelates to molecules, preferably proteins, more preferablyimmunoglobulins, that comprise an IgG constant domain, or FcRn bindingfragment thereof (preferably a Fc or hinge-Fc domain fragment), havingone or more amino acid modifications (i.e., substitutions, insertions ordeletions) in one or more regions that interact with the FcRn, whichmodifications increase the affinity of the IgG or fragment thereof, forthe FcRn, and also increase the in vivo half-life of the molecule. Inpreferred embodiments, the one or more amino acid modifications are madein one or more of residues 251-256, 285-290, 308-314, 385-389, and428-436 of the IgG hinge-Fc region (for example, as in the human IgG1hinge-Fc region depicted in FIG. 4, SEQ ID NO:83), or analogous residuesthereof, as determined by amino acid sequence alignment, in other IgGhinge-Fc regions. In a preferred embodiment, the amino acidmodifications are made in a human IgG constant domain, or FcRn-bindingdomain thereof. In a certain embodiment, the modifications are not madeat residues 252, 254, or 256 (i.e., all are made at one or more ofresidues 251, 253, 255, 285-290, 308-314, 385-389, or 428-436) of theIgG constant domain. In a more preferred embodiment, the amino acidmodifications are not the substitution with leucine at residue 252, withserine at 254, and/or with phenylalanine at position 256. In particular,in preferred embodiments, such modifications are not made when the IgGconstant domain, hinge-Fc domain, hinge-Fc domain or other FcRn-bindingfragment thereof is derived from a mouse.

[0049] The amino acid modifications may be any modification, preferablyat one or more of residues 251-256, 285-290, 308-314, 385-389, and428-436, that increases the in vivo half-life of the IgG constantdomain, or FcRn-binding fragment thereof (e.g., Fc or hinge-Fc domain),and any molecule attached thereto, and increases the affinity of theIgG, or fragment thereof, for FcRn. Preferably, the one or moremodifications also result in a higher binding affinity of the constantdomain, or FcRn-binding fragment thereof, for FcRn at pH 6.0 than at pH7.4. In other embodiments, the modifications alter (i.e., increase ordecrease) bioavailability of the molecule, in particular, alters (i.e.,increases or decreases) transport (or concentration or half-life) of themolecule to mucosal surfaces (e.g., of the lungs) or other portions of atarget tissue. In a preferred embodiment, the amino acid modificationsalter (preferably, increase) transport or concentration or half-life ofthe molecule to the lungs. In other embodiments, the amino acidmodifications alter (preferably, increase) transport (or concentrationor half-life) of the molecule to the heart, pancreas, liver, kidney,bladder, stomach, large or small intestine, respiratory tract, lymphnodes, nervous tissue (central and/or peripheral nervous tissue),muscle, epidermis, bone, cartilage, joints, blood vessels, bone marrow,prostate, ovary, uterine, tumor or cancer tissue, etc. In a preferredembodiment, the amino acid modifications do not abolish, or, morepreferably, do not alter, other immune effector or receptor bindingfunctions of the constant domain, for example, but not limited tocomplement fixation, ADCC and binding to FcγRI, FcγRII, and FcγRIII, ascan be determined by methods well-known and routine in the art. Inanother preferred embodiment, the modified FcRn binding fragment of theconstant domain does not contain sequences that mediate immune effectorfunctions or other receptor binding. Such fragments may be particularlyuseful for conjugation to a non-IgG or non-immunoglobulin molecule toincrease the in vivo half-life thereof. In yet another embodiment, theeffector functions are selectively altered (e.g., to reduce or increaseeffector functions).

[0050] In preferred embodiments, the amino acid modifications aresubstitutions at one or more of residues 308, 309, 311, 312 and 314,particularly a substitution with threonine at position 308, proline atposition 309, serine at position 311, aspartic acid at position 312,and/or leucine at position 314. Alternatively, the modification is thesubstitution with an isoleucine at position 308, proline at position309, and/or a glutamic acid at position 311. In yet another embodiment,residues at one or more of positions 308, 309, 311, 312, and 314, aresubstituted with threonine, proline, leucine, alanine, and alanine,respectively. Accordingly, in certain embodiments the residue atposition 308 is substituted with threonine or isoleucine, the residue atposition 309 is substituted with proline, the residue at position 311 issubstituted with serine, glutamic acid or leucine, the residue atposition 312 is substituted with alanine, and/or the residue at position314 is substituted with leucine or alanine. In a preferred embodiment,the substitution is a threonine at position 308, a proline at position309, a serine at position 311, an aspartic acid at position 312, and/ora leucine at position 314.

[0051] In preferred embodiments, the amino acid modifications aresubstitutions at one or more of residues 251, 252, 254, 255, and 256. Inspecific embodiments, residue 251 is substituted with leucine orarginine, residue 252 is substituted with tyrosine, phenylalanine,serine, tryptophan or threonine, residue 254 is substituted withthreonine or serine, residue 255 is substituted with arginine, leucine,glycine, or isoleucine, and/or residue 256 is substituted with serine,arginine, glutamine, glutamic acid, aspartic acid, alanine, asparagineor threonine. In a more specific embodiment, residue 251 is substitutedwith leucine, residue 252 is substituted with tyrosine, residue 254 issubstituted with threonine or serine, residue 255 is substituted witharginine, and/or residue 256 is substituted with glutamic acid.

[0052] In preferred embodiments, the amino acid modifications aresubstitutions at one or more of residues 428, 433, 434, and 436. Inspecific embodiments, residue 428 is substituted with threonine,methionine, leucine, phenylalanine, or serine, residue 433 issubstituted with lysine, arginine, serine, isoleucine, proline,glutamine or histidine, residue 434 is substituted with phenylalanine,tyrosine, or histidine, and/or residue 436 is substituted withhistidine, asparagine, arginine, threonine, lysine, or methionine. In amore specific embodiment, residues at position 428 and/or 434 aresubstituted with methionine, and/or histidine respectively.

[0053] In preferred embodiments, the amino acid modifications aresubstitutions at one or more of residues 385, 386, 387, and 389, morespecifically, having substitutions at one or more of these positions. Inspecific embodiments, residue 385 is substituted with arginine, asparticacid, serine, threonine, histidine, lysine, alanine or glycine, residue386 is substituted with threonine, proline, aspartic acid, serine,lysine, arginine, isoleucine, or methionine, residue 387 is substitutedwith arginine, proline, histidine, serine, threonine, or alanine, and/orresidue 389 is substituted with proline, serine or asparagine. In morespecific embodiments, residues at one or more positions 385, 386, 387,and 389 are substituted with arginine, threonine, arginine, and proline,respectively. In yet another specific embodiment, residues at one ormore positions 385, 386, and 389 are substituted with aspartic acid,proline, and serine, respectively.

[0054] In particular embodiments, amino acid modifications are made atone or a combination of residues 251, 252, 254, 255, 256, 308, 309, 311,312, 314, 385, 386, 387, 389, 428, 433, 434, and/or 436, particularlywhere the modifications are one or more of the amino acid substitutionsdescribed immediately above for these residues.

[0055] In a preferred embodiment, the molecule of the invention containsa Fc region, or FcRn-binding domain thereof, having one or more of thefollowing substitutions: leucine at residue 251, tyrosine at residue252, threonine or serine at residue 254, arginine at residue 255,threonine at residue 308, proline at residue 309, serine at residue 311,aspartic acid at residue 312, leucine at residue 314, arginine atresidue 385, threonine at residue 386, arginine at residue 387, prolineat residue 389, methionine at residue 428, and/or tyrosine at residue434.

[0056] In a preferred embodiment, the FcRn binding domain has asubstitution at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16 or all 18 ofresidues 251, 252, 254, 255, 256, 308, 309, 311, 312, 314, 385, 386,387, 389, 428, 433, 434, and/or 436.

[0057] Amino acid modifications can be made by any method known in theart and many such methods are well known and routine for the skilledartisan. For example, but not by way of limitation, amino acidsubstitutions, deletions and insertions may be accomplished using anywell-known PCR-based technique. Amino acid substitutions may be made bysite-directed mutagenesis (see, for example, Zoller and Smith, Nucl.Acids Res. 10:6487-6500, 1982; Kunkel, Proc. Natl. Acad. Sci USA 82:488,1985, which are hereby incorporated by reference in their entireties).Mutants that result in increased affinity for FcRn and increased in vivohalf-life may readily be screened using well-known and routine assays,such as those described in Section 5.11, infra. In a preferred method,amino acid substitutions are introduced at one or more residues in theIgG constant domain or FcRn-binding fragment thereof and the mutatedconstant domains or fragments are expressed on the surface ofbacteriophage which are then screened for increased FcRn bindingaffinity (see, in particular, Section 5.2 and 5.11, infra).

[0058] Preferably, the amino acid residues to be modified are surfaceexposed residues. Additionally, in making amino acid substitutions,preferably the amino acid residue to be substituted is a conservativeamino acid substitution, for example, a polar residue is substitutedwith a polar residue, a hydrophilic residue with a hydrophilic residue,hydrophobic residue with a hydrophobic residue, a positively chargedresidue with a positively charged residue, or a negatively chargedresidue with a negatively charged residue. Moreover, preferably, theamino acid residue to be modified is not highly or completely conservedacross species and/or is critical to maintain the constant domaintertiary structure or to FcRn binding. For example, but not by way oflimitation, modification of the histidine at residue 310 is notpreferred.

[0059] Specific mutants of the Fc domain that have increased affinityfor FcRn were isolated after the third-round panning (as described inSection 6) from a library of mutant human IgG1 molecules havingmutations at residues 308-314 (histidine at position 310 and tryptophanat position 313 are fixed), those isolated after the fifth-round panningof the library for residues 251-256 (isoleucine at position 253 isfixed), those isolated after fourth-round panning of the library forresidues 428-436 (histidine at position 429, glutamic acid at position430, alanine at position 431, leucine at position 432, and histidine atposition 435 are fixed), and those isolated after sixth-round panning ofthe library for residues 385-389 (glutamic acid at position 388 isfixed) are listed in Table I. The wild type human IgG1 has a sequenceVal-Leu-His-Gln-Asp-Trp-Leu (SEQ ID NO:86) at positions 308-314,Leu-Met-Ile-Ser-Arg-Thr (SEQ ID NO:87) at positions 251-256,Met-His-Glu-Ala-Leu-His-Asn-His-Tyr (SEQ ID NO:88) at positions 428-436,and Gly-Gln-Pro-Glu-Asn (SEQ ID NO:89) at positions 385-389. TABLE IMUTANTS ISOLATED BY PANNING LIBRARY MUTANTS* 251-256Leu Tyr Ile Thr Arg Glu  (SEQ ID NO:90) Leu

Tyr  Ile Ser Arg Thr (SEQ ID NO:91) Leu

Tyr  Ile Ser Arg

Ser (SEQ ID NO:92) Leu

Tyr  Ile Ser Arg

Arg (SEQ ID NO:93) Leu

Tyr  Ile Ser Arg

Gln (SEQ ID NO:94) Leu

Trp  Ile Ser Arg Thr (SEQ ID NO:95) Leu Tyr Ile Ser Leu Gln (SEQ IDNO:96) Leu Phe Ile Ser Arg Asp (SEQ ID NO:97) Leu Phe Ile Ser Arg Thr(SEQ ID NO:98) Leu Phe Ile Ser Arg Arg (SEQ ID NO:99) Leu Phe Ile ThrGly Ala (SEQ ID NO: 100) Leu Ser Ile Ser Arg Glu (SEQ ID NO:101) 308-314Thr Pro His Ser Asp Trp Leu (SEQ ID NO:103) Ile Pro His Glu Asp Trp Leu(SEQ ID NO:104) 385-389 Arg Thr Arg Glu Pro (SEQ ID NO:105)

Asp 

Pro Pro Glu

Ser (SEQ ID NO:106) Ser Asp Pro Glu Pro (SEQ ID NO:107) Thr Ser His GluAsn (SEQ ID NO:108) Ser Lys Ser Glu Asn (SEQ ID NO:109) His Arg Ser GluAsn (SEQ ID NO:110) Lys Ile Arg Glu Asn (SEQ ID NO:111) Gly Ile Thr GluSer (SEQ ID NO:112) Ser Met Ala Glu Pro (SEQ ID NO:113) 428-436 Met HisGlu Ala Leu

Arg 

Tyr His

His (SEQ ID NO:114) Met His Glu Ala Leu His Phe His His (SEQ ID NO:115)Met His Glu Ala Leu Lys Phe His His (SEQ ID NO:116) Met His Glu Ala LeuSer Tyr His Arg (SEQ ID NO:117) Thr His Glu Ala Leu His Tyr His Thr (SEQID NO:118) Met His Glu Ala Leu His Tyr His Tyr (SEQ ID NO:119)

[0060] The underlined sequences in Table I correspond to sequences thatoccurred 10 to 20 times in the final round of panning and the sequencesin italics correspond to sequences that occurred 2 to 5 times in thefinal round of panning. Those sequences that are neither underlined noritalicized occurred once in the final round of panning.

[0061] In one preferred embodiment, the invention provides modifiedimmunoglobulin molecules (e.g., various antibodies) that have increasedin vivo half-life and affinity for FcRn relative to unmodified molecules(and, in preferred embodiments, altered bioavailabilty such as increasedor decreased transport to mucosal surfaces or other target tissues).Such immunoglobulin molecules include IgG molecules that naturallycontain an FcRn binding domain and other non-IgG immunoglobulins (e.g.,IgE, IgM, IgD, IgA and IgY) or fragments of immunoglobulins that havebeen engineered to contain an FcRn-binding fragment (i. e., fusionproteins comprising non-IgG immunoglobulin or a portion thereof and anFcRn binding domain). In both cases the FcRn-binding domain has one ormore amino acid modifications that increase the affinity of the constantdomain fragment for FcRn.

[0062] The modified immunoglobulins include any immunoglobulin moleculethat binds (preferably, immunospecifically, i.e., competes offnon-specific binding), as determined by immunoassays well known in theart for assaying specific antigen-antibody binding) an antigen andcontains an FcRn-binding fragment. Such antibodies include, but are notlimited to, polyclonal, monoclonal, bi-specific, multi-specific, human,humanized, chimeric antibodies, single chain antibodies, Fab fragments,F(ab′)₂ fragments, disulfide-linked Fvs, and fragments containing eithera VL or VH domain or even a complementary determining region (CDR) thatspecifically binds an antigen, in certain cases, engineered to containor fused to an FcRn binding domain.

[0063] The IgG molecules of the invention, and FcRn-binding fragmentsthereof, are preferably IgG1 subclass of IgGs, but may also be any otherIgG subclasses of given animals. For example, in humans, the IgG classincludes IgG1, IgG2, IgG3, and IgG4; and mouse IgG includes IgG1, IgG2a,IgG2b, IgG2c and IgG3. It is known that certain IgG subclasses, forexample, mouse IgG2b and IgG2c, have higher clearance rates than, forexample, IgG1 (Medesan et al., Eur. J. Immunol., 28:2092-2100, 1998).Thus, when using IgG subclasses other than IgG1, it may be advantageousto substitute one or more of the residues, particularly in the CH2 andCH3 domains, that differ from the IgG1 sequence with those of IgG1,thereby increasing the in vivo half-life of the other types of IgG.

[0064] The immunoglobulins (and other proteins used herein) may be fromany animal origin including birds and mammals. Preferably, theantibodies are human, rodent (e.g., mouse and rat), donkey, sheep,rabbit, goat, guinea pig, camel, horse, or chicken. As used herein,“human” antibodies include antibodies having the amino acid sequence ofa human immunoglobulin and include antibodies isolated from humanimmunoglobulin libraries or from animals transgenic for one or morehuman immunoglobulin and that do not express endogenous immunoglobulins,as described infra and, for example, in U.S. Pat. No. 5,939,598 byKucherlapati et al.

[0065] The antibodies of the present invention may be monospecific,bispecific, trispecific or of greater multispecificity. Multispecificantibodies may be specific for different epitopes of a polypeptide ormay be specific for heterologous epitopes, such as a heterologouspolypeptide or solid support material. See, e.g., PCT publications WO93/17715, WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al., J.Immunol., 147:60-69, 1991; U.S. Pat. Nos. 4,474,893; 4,714,681;4,925,648; 5,573,920; 5,601,819; Kostelny et al., J. Immunol.,148:1547-1553, 1992.

[0066] The antibodies of the invention include derivatives that areotherwise modified, i.e., by the covalent attachment of any type ofmolecule to the antibody such that covalent attachment does not preventthe antibody from binding antigen and/or generating an anti-idiotypicresponse. For example, but not by way of limitation, the antibodyderivatives include antibodies that have been modified, e.g., byglycosylation, acetylation, pegylation, phosphorylation, amidation,derivatization by known protecting/blocking groups, proteolyticcleavage, linkage to a cellular ligand or other protein, etc. Any ofnumerous chemical modifications may be carried out by known techniques,including, but not limited to, specific chemical cleavage, acetylation,formylation, metabolic synthesis of tunicamycin, etc. Additionally, thederivative may contain one or more non-classical amino acids.

[0067] Monoclonal antibodies can be prepared using a wide variety oftechniques known in the art including the use of hybridoma, recombinant,and phage display technologies, or a combination thereof. For example,monoclonal antibodies can be produced using hybridoma techniquesincluding those known in the art and taught, for example, in Harlow etal., Antibodies: A Laboratory Manual, (Cold Spring Harbor LaboratoryPress, 2nd ed. 1988); Hammerling, et al., in: Monoclonal Antibodies andT-Cell Hybridomas, pp. 563-681 (Elsevier, N.Y., 1981) (both of which areincorporated herein by reference in their entireties). The term“monoclonal antibody” as used herein is not limited to antibodiesproduced through hybridoma technology. The term “monoclonal antibody”refers to an antibody that is derived from a single clone, including anyeukaryotic, prokaryotic, or phage clone, and not the method by which itis produced.

[0068] Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well known in the art. In anon-limiting example, mice can be immunized with an antigen of interestor a cell expressing such an antigen. Once an immune response isdetected, e.g., antibodies specific for the antigen are detected in themouse serum, the mouse spleen is harvested and splenocytes isolated. Thesplenocytes are then fused by well known techniques to any suitablemyeloma cells. Hybridomas are selected and cloned by limiting dilution.The hybridoma clones are then assayed by methods known in the art forcells that secrete antibodies capable of binding the antigen. Ascitesfluid, which generally contains high levels of antibodies, can begenerated by inoculating mice intraperitoneally with positive hybridomaclones.

[0069] Antibody fragments which recognize specific epitopes may begenerated by known techniques. For example, Fab and F(ab′)₂ fragmentsmay be produced by proteolytic cleavage of immunoglobulin molecules,using enzymes such as papain (to produce Fab fragments) or pepsin (toproduce F(ab′)₂ fragments). F(ab′)₂ fragments contain the complete lightchain, and the variable region, the CH1 region and the hinge region ofthe heavy chain.

[0070] For example, antibodies can also be generated using various phagedisplay methods known in the art. In phage display methods, functionalantibody domains are displayed on the surface of phage particles whichcarry the polynucleotide sequences encoding them. In a particularembodiment, such phage can be utilized to display antigen bindingdomains, such as Fab and Fv or disulfide-bond stabilized Fv, expressedfrom a repertoire or combinatorial antibody library (e.g., human ormurine). Phage expressing an antigen binding domain that binds theantigen of interest can be selected or identified with antigen, e.g.,using labeled antigen or antigen bound or captured to a solid surface orbead. Phage used in these methods are typically filamentous phage,including fd and M13. The antigen binding domains are expressed as arecombinantly fused protein to either the phage gene III or gene VIIIprotein. Alternatively, the modified FcRn binding portion ofimmunoglobulins of the present invention can be also expressed in aphage display system. Examples of phage display methods that can be usedto make the immunoglobulins, or fragments thereof, of the presentinvention include those disclosed in Brinkman et al., J. Immunol.Methods, 182:41-50, 1995; Ames et al., J. Immunol. Methods, 184:177-186,1995; Kettleborough et al., Eur. J. Immunol., 24:952-958, 1994; Persicet al., Gene, 187:9-18, 1997; Burton et al., Advances in Immunology,57:191-280, 1994; PCT application No. PCT/GB91/01134; PCT publicationsWO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/1 1236; WO95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409;5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698;5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108;each of which is incorporated herein by reference in its entirety.

[0071] As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired fragments, and expressed in any desired host, includingmammalian cells, insect cells, plant cells, yeast, and bacteria, e.g.,as described in detail below. For example, techniques to recombinantlyproduce Fab, Fab′ and F(ab′)₂ fragments can also be employed usingmethods known in the art such as those disclosed in PCT publication WO92/22324; Mullinax et al., BioTechniques, 12(6):864-869, 1992; and Sawaiet al., AJRI, 34:26-34, 1995; and Better et al., Science, 240:1041-1043,1988 (each of which is incorporated by reference in its entirety).Examples of techniques which can be used to produce single-chain Fvs andantibodies include those described in U.S. Pat. Nos. 4,946,778 and5,258,498; Huston et al., Methods in Enzymology, 203:46-88, 1991; Shu etal., PNAS, 90:7995-7999, 1993; and Skerra et al., Science,240:1038-1040, 1988.

[0072] For some uses, including in vivo use of antibodies in humans andin vitro detection assays, it may be preferable to use chimeric,humanized, or human antibodies. A chimeric antibody is a molecule inwhich different portions of the antibody are derived from differentanimal species, such as antibodies having a variable region derived froma murine monoclonal antibody and a constant region derived from a humanimmunoglobulin. Methods for producing chimeric antibodies are known inthe art. See e.g., Morrison, Science, 229:1202, 1985; Oi et al.,BioTechniques, 4:214 1986; Gillies et al., J. Immunol. Methods,125:191-202, 1989; U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816,397,which are incorporated herein by reference in their entireties.Humanized antibodies are antibody molecules from non-human species thatbind the desired antigen having one or more complementarity determiningregions (CDRs) from the non-human species and framework regions from ahuman immunoglobulin molecule. Often, framework residues in the humanframework regions will be substituted with the corresponding residuefrom the CDR donor antibody to alter, preferably improve, antigenbinding. These framework substitutions are identified by methods wellknown in the art, e.g., by modeling of the interactions of the CDR andframework residues to identify framework residues important for antigenbinding and sequence comparison to identify unusual framework residuesat particular positions. See, e.g., Queen et al., U.S. Pat. No.5,585,089; Riechmann et al., Nature, 332:323, 1988, which areincorporated herein by reference in their entireties. Antibodies can behumanized using a variety of techniques known in the art including, forexample, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S.Pat. Nos. 5,225,539; 5,530,101 and 5,585,089), veneering or resurfacing(EP 592,106; EP 519,596; Padlan, Molecular Immunology, 28(4/5):489-498,1991; Studnicka et al., Protein Engineering, 7(6):805-814, 1994; Roguskaet al., Proc Natl. Acad. Sci. USA, 91:969-973, 1994), and chainshuffling (U.S. Pat. No. 5,565,332), all of which are herebyincorporated by reference in their entireties.

[0073] Completely human antibodies are particularly desirable fortherapeutic treatment of human patients. Human antibodies can be made bya variety of methods known in the art including phage display methodsdescribed above using antibody libraries derived from humanimmunoglobulin sequences. See U.S. Pat. Nos. 4,444,887 and 4,716,111;and PCT publications WO 98/46645; WO 98/50433; WO 98/24893; WO 98/16654;WO 96/34096; WO 96/33735; and WO 91/10741, each of which is incorporatedherein by reference in its entirety.

[0074] Human antibodies can also be produced using transgenic mice whichare incapable of expressing functional endogenous immunoglobulins, butwhich can express human immunoglobulin genes. For an overview of thistechnology for producing human antibodies, see Lonberg and Huszar, Int.Rev. Immunol., 13:65-93, 1995. For a detailed discussion of thistechnology for producing human antibodies and human monoclonalantibodies and protocols for producing such antibodies, see, e.g., PCTpublications WO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735;European Patent No.0 598 877; U.S. Pat. Nos. 5,413,923; 5,625,126;5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793;5,916,771; and 5,939,598, which are incorporated by reference herein intheir entireties. In addition, companies such as Abgenix, Inc.(Freemont, Calif.), Medarex (NJ) and Genpharm (San Jose, Calif.) can beengaged to provide human antibodies directed against a selected antigenusing technology similar to that described above.

[0075] Completely human antibodies which recognize a selected epitopecan be generated using a technique referred to as “guided selection.” Inthis approach a selected non-human mono clonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. (Jespers et al., Bio/technology,12:899-903, 1988).

[0076] In particular embodiments, the modified antibodies have in vivotherapeutic and/or prophylactic uses. Examples of therapeutic andprophylactic antibodies which may be so modified include, but are notlimited to, SYNAGIS® (MedImmune, MD) which is a humanizedanti-respiratory syncytial virus (RSV) monoclonal antibody for thetreatment of patients with RSV infection; HERCEPTIN® (Trastuzumab)(Genentech, CA) which is a humanized anti-HER2 monoclonal antibody forthe treatment of patients with metastatic breast cancer; REMICADE®(infliximab) (Centocor, PA) which is a chimeric anti-TNFα monoclonalantibody for the treatment of patients with Crone's disease; REOPRO®(abciximab) (Centocor) which is an anti-glycoprotein IIb/IIIa receptoron the platelets for the prevention of clot formation; ZENAPAX®(daclizumab) (Roche Pharmaceuticals, Switzerland) which is animmunosuppressive, humanized anti-CD25 monoclonal antibody for theprevention of acute renal allograft rejection. Other examples are ahumanized anti-CD 18 F(ab′)₂ (Genentech); CDP860 which is a humanizedanti-CD18 F(ab′)₂ (Celltech, UK); PRO542 which is an anti-HIV gp120antibody fused with CD4 (Progenics/Genzyme Transgenics); Ostavir whichis a human anti Hepatitis B virus antibody (Protein DesignLab/Novartis); PROTOVIR™ which is a humanized anti-CMV IgG1 antibody(Protein Design Lab/Novartis); MAK-195 (SEGARD) which is a murineanti-TNF-α F(ab′)₂ (Knoll Pharma/BASF); IC14 which is an anti-CD14antibody (ICOS Pharm); a humanized anti-VEGF IgG1 antibody (Genentech);OVAREX™ which is a murine anti-CA 125 antibody (Altarex); PANOREX™ whichis a murine anti-17-IA cell surface antigen IgG2a antibody (GlaxoWellcome/Centocor); BEC2 which is a murine anti-idiotype (GD3 epitope)IgG antibody (ImClone System); IMC-C225 which is a chimeric anti-EGFRIgG antibody (ImClone System); VITAXIN™ which is a humanized anti-αVβ3integrin antibody (Applied Molecular Evolution/MedImmune); Campath1H/LDP-03 which is a humanized anti CD52 IgG1 antibody (Leukosite);Smart M195 which is a humanized anti-CD33 IgG antibody (Protein DesignLab/Kanebo); RITUXANT™ which is a chimeric anti-CD20 IgG1 antibody (IDECPharm/Genentech, Roche/Zettyaku); LYMPHOCIDE™ which is a humanizedanti-CD22 IgG antibody (Immunomedics); Smart ID10 which is a humanizedanti-HLA antibody (Protein Design Lab); ONCOLYM™ (Lym-1) is aradiolabelled murine anti-HLA DIAGNOSTIC REAGENT antibody (Techniclone);ABX-IL8 is a human anti-IL8 antibody (Abgenix); anti-CD11a is ahumanized IgG1 antibody (Genetech/Xoma); ICM3 is a humanized anti-ICAM3antibody (ICOS Pharm); IDEC-114 is a primatized anti-CD80 antibody (IDECPharm/Mitsubishi); ZEVALIN™ is a radiolabelled murine anti-CD20 antibody(IDEC/Schering AG); IDEC-131 is a humanized anti-CD40L antibody(IDEC/Eisai); IDEC-151 is a primatized anti-CD4 antibody (IDEC);IDEC-152 is a primatized anti-CD23 antibody (IDEC/Seikagaku); SMARTanti-CD3 is a humanized anti-CD3 IgG (Protein Design Lab); 5G1.1 is ahumanized anti-complement factor 5 (C5) antibody (Alexion Pharm); D2E7is a humanized anti-TNF-α antibody (CAT/BASF); CDP870 is a humanizedanti-TNF-α Fab fragment (Celltech); IDEC-151 is a primatized anti-CD4IgG1 antibody (IDEC Pharm/SmithKline Beecham); MDX-CD4 is a humananti-CD4 IgG antibody (Medarex/Eisai/Genmab); CDP571 is a humanizedanti-TNF-α IgG4 antibody (Celltech); LDP-02 is a humanized anti-α4β7antibody (LeukoSite/Genentech); OrthoClone OKT4A is a humanized anti-CD4IgG antibody (Ortho Biotech); ANTOVA™ is a humanized anti-CD40L IgGantibody (Biogen); ANTEGREN™ is a humanized anti-VLA-4 IgG antibody(Elan); MDX-33 is a human anti-CD64 (FcγR) antibody (Medarex/Centeon);SCH55700 is a humanized anti-IL-5 IgG4 antibody (Celltech/Schering);SB-240563 and SB-240683 are humanized anti-IL-5 and IL-4 antibodies,respectively, (SmithKline Beecham); rhuMab-E25 is a humanized anti-IgEIgG1 antibody (Genentech/Norvartis/Tanox Biosystems); IDEC-152 is aprimatized anti-CD23 antibody (IDEC Pharm); ABX-CBL is a murine antiCD-147 IgM antibody (Abgenix); BTI-322 is a rat anti-CD2 IgG antibody(Medimmune/Bio Transplant); Orthoclone/OKT3 is a murine anti-CD3 IgG2aantibody (ortho Biotech); SIMULECT™ is a chimeric anti-CD25 IgG1antibody (Novartis Pharm); LDP-01 is a humanized anti-β₂-integrin IgGantibody (LeukoSite); Anti-LFA-1 is a murine anti CD18 F(ab′)₂(Pasteur-Merieux/Immunotech); CAT-152 is a human anti-TGF-β₂ antibody(Cambridge Ab Tech); and Corsevin M is a chimeric anti-Factor VIIantibody (Centocor).

[0077] In specific embodiments, the invention provides modifiedantibodies having one or more of the mutations described herein and thatimmunospecifically bind RSV, e.g., SYNAGIS®. The present invention alsoprovides modified antibodies having one or more of the mutationsdescribed herein and that comprise a variable heavy (VH) and/or variablelight (VL) domain having the amino acid sequence of any VH and/or VLdomain listed in Table III. The present invention further encompassesanti-RSV antibodies comprising one or more VH complementaritydetermining regions (CDRs) and/or one or more VL CDRs having the aminoacid sequence of one or more VH CDRs and/or VL CDRS listed in Table IIIor one or more of the CDRs listed in Table II wherein one or more of thebolded and underlined residues has an amino acid substitution,preferably that increases the affinity of the antibody for RSV. Inspecific embodiments, the antibody to be modified is AFFF, p12f2, p12f4,p11d4, Ale109, A12a6, A13c4, A17d4, A4B4, A8C7, 1X-493L1FR, H3-3F4,M3H9, Y10H6, DG, AFFF(1), 6H8, L1-7E5, L215B10, A13A11, A1H5, A4B4(1),A4B4L1FR-S28R, A4B4-F52S. TABLE II CDR Sequences of SYNAGIS ® CDRSequence SEQ ID NO: VH1 T S GMSVG 1 VH2 DIWWD D DK KD YNPSLK S 2 VH3 SMI T N W YFDV 3 VL1 KCQLS VGYMH 4 VL2 DT SKLA S 5 VL3 FQGS G YP F T 6

[0078] TABLE III ANTI-RSV ANTIBODIES Antibody Name VH Domain VH CDR1 VHCDR2 VH CDR3 VL Domain VL CDR1 VL CDR2 VL CDR3 SYNAGIS SEQ ID NO:7TSGMSVG (SEQ ID NO:1) DIWWDDKKDYNPSLKS SMITNWYFDV SEQ ID NO:8 KCQLSVGYMHDTSKLAS FQGSGYPFT (SEQ ID NO:2) (SEQ ID NO:3) (SEQ ID NO:4) (SEQ IDNO:5) (SEQ ID NO:6) AFFF SEQ ID NO:9 T A GMSVG (SEQ ID NO:10)DIWWDDKKDYNPSLKS SMITN F YFDV SEQ ID NO:13 +E,usn SASSSVGYMH DT F KLASFQ FS GYPFT (SEQ ID NO:2) (SEQ ID NO:12) (SEQ ID NO:14) (SEQ ID NO 15)(SEQ ID NO:16) p12f2 SEQ ID NO:17 T P GMSVG (SEQ ID NO.18) DIWWDDKK HYNPSLK D D MI F N F YFDV SEQ ID NO.21 e,uns SLSSRVGYMH DT FY L S SFQGSGYPFT (SEQ ID NO:19) (SEQ ID NO:20) (SEQ ID NO:22) (SEQ ID NO:23)(SEQ ID NO:6) p12f4 SEQ ID NO:24 T P GMSVG (SEQ ID NO.18) DIWWD G KK HYNPSLK D D MI F N F YFDV SEQ ID NO.26 SLSSR VGYMH DT RG L P S FQSGYPFT(SEQ ID NO:25) (SEQ ID NO:20) (SEQ ID NO:22) (SEQ ID NO:27) (SEQ IDNO.6) p11d4 SEQ ID NO.28 T P GMSVG (SEQ ID NO:18) DIWWD G KK H YNPSLK DD MIFNWYFDV SEQ ID NO:30 SPSSR VGYMH DT MR LAS FQGSGYPFT (SEQ ID NO:25)(SEQ ID NO:29) (SEQ ID NO:31) (SEQ ID NO:32) (SEQ ID NO:6) Ale109 SEQ IDNO.33 T A GMSVG (SEQ ID NO:10) DIWWD G KK H YNPSLK D D MI F NWYFDV SEQID NO:34 SLSSR VGYMH DT F KL S S FQGSGYPFT (SEQ ID NO:25) (SEQ ID NO:29)(SEQ ID NO:22) (SEQ ID NO.35) (SEQ ID NO:6) A12a6 SEQ ID NO:36 T A GMSVG(SEQ ID NO:10) DIWWD G KKDYNPSLK D D MI F N F YFDV SEQ ID NO 38 SASSRVGYMH DT F KL S S FQGSGYPFT (SEQ ID NO:37) (SEQ ID NO.20) (SEQ ID NO:39)(SEQ ID NO:35) (SEQ ID NO:6) A13c4 SEQ ID NO:40 T A GMSVG (SEQ ID NO:10)DIWWD G KK S YNPSLK D D MI F N F YFDV SEQ ID NO:42 SLSSR VGYMH DT MYQS SFQGSGYPFT (SEQ ID NO:41) (SEQ IDNO:20) (SEQ ID NO:22) (SEQ ID NO:43)(SEQ ID NO:6) A17d4 SEQ ID NO:44 T A GMSVG (SEQ ID NO:10) DIWWDDKK SYNPSLK D D MI F N F YFDV SEQ ID NO:46 LPSSR VGYMH DT MYQS S FQOSGYPFT(SEQ ID NO.45) (SEQ ID NO.20) (SEQ ID NO.47) (SEQ ID NO:43) (SEQ IDNO:6) A4B4 SEQ ID NO.48 T A GMSVG (SEQ ID NO:10) DIWWDDKK H YNPSLK D DMI F N F YFDV SEQ ID NO:49 SASSR VGYMH DT FF L D S FQGSGYPFT (SEQ IDNO:19) (SEQ ID NO:20) (SEQ ID NO:39) (SEQ ID NO:50) (SEQ ID NO:6) A8C7SEQ ID NO:51 T A GMSVG (SEQ ID NO:10) DIWWDDKK S YNPSLK D D MI F NWYFDVSEQ ID NO:52 SPSSR VGYMH DT RYQS S FQGSGYPFT (SEQ ID NO.45) (SEQ IDNO:29) (SEQ ID NO:31) (SEQ ID NO:53) (SEQ ID NO:6) 1X-493L1FR SEQ IDNO:7 TSGMSVG (SEQ ID NO:1) DIWWDDKKDYNPSLKS SMITNWYFDV SEQ ID NO:54 SASSSVGYMH DTSKLAS FQGSGYPFT (SEQ ID NO:2) (SEQ ID NO:3) (SEQ ID NO:14) (SEQID NO:5) (SEQ ID NO:6) H3-3F4 SEQ ID NO:55 T A GMSVG (SEQ ID NO:10)DIWWDDKKDYNPSLKS D MI F NWYFDV SEQ ID NO:56 SASS SVGYMH DT F KLASFQGSGYPFT (SEQ ID NO:2) (SEQ ID NO:29) (SEQ ID NO:14) (SEQ ID NO:15)(SEQ ID NO:6) M3H9 SEQ ID NO:55 T A GMSVG (SEQ ID NO:10)DIWWDDKKDYNPSLKS D MI F NWYFDV SEQ ID NO:56 SASS SVGYMH DT Y K QT SFQGSGYPFT (SEQ ID NO:2) (SEQ ID NO:29) (SEQ ID NO:14) (SEQ ID NO:57)(SEQ ID NO:6) Y10H6 SEQ ID NO:55 T A GMSVG (SEQ ID NO:10)DIWWDDKKDYNPSLKS D MI F NWYFDV SEQ ID NO:58 SASS SVGYMH DT RY L S SFQGSGYPFT (SEQ ID NO 2) (SEQ ID NO:29) (SEQ ID NO:14) (SEQ ID NO.59)(SEQ ID NO.6) DG SEQ ID NO 78 T A GMSVG (SEQ ID NO:10) DIWWDDKKDYNPSLKSD MITN F YFDV SEQ ID NO:56 SASS SVGYMH DT F KLAS FQGSGYPFT (SEQ ID NO:2)(SEQ ID NO.79) (SEQ ID NO:14) (SEQ ID NO:15) (SEQ ID NO:6) AFFF(1) SEQID NO:9 T A GMSVG (SEQ ID NO:10) DIWWDDKKDYNPSLKS S MITN F YFDV SEQ IDNO:60 SASS SVGYMH DT F KLAS FQGS F YPFT (SEQ ID NO:2) (SEQ ID NO:12)(SEQ ID NO:14) (SEQ ID NO:15) (SEQ ID NO:61) 6H8 SEQ ID NO:78 T A GMSVG(SEQ ID NO:10) DIWWDDKKDYNPSLKS D MITN F YFDV SEQ ID NO:62 SASS SVGYMHDT F KL T S FQGSGYPFT (SEQ ID NO:2) (SEQ ID NO.79) (SEQ ID NO:14) (SEQID NO.63) (SEQ ID NO:6) L1-7E5 SEQ ID NO:78 T A GMSVG (SEQ ID NO:10)DIWWDDKKDYNPSLKS D MITN F YFDV SEQ ID NO:64 SASSR VGYMH DT F KLASFQGSGYPFT (SEQ ID NO.2) (SEQ ID NO:79) (SEQ ID NO:39) (SEQ ID NO.15)(SEQ ID NO:6) L215B10 SEQ ID NO:78 T A GMSVG (SEQ ID NO:10)DIWWDDKKDYNPSLKS D MITN F YFDV SEQ ID NO:65 SASS SVGYMH DT FR LASFQGSGYPFT (SEQ ID NO.2) (SEQ ID NO:79) (SEQ ID NO:14) (SEQIDNO.66) (SEQID NO:66) (SEQ ID NO:6) A13A11 SEQ ID NO:67 T A GMSVG (SEQ ID NO:10)DIWWDDKK H YNPSLK D D MI F NWYFDV SEQ ID NO:68 SPSSR VGYMH DT YRHS SFQGSGYPFT (SEQ ID NO:19) (SEQ ID NO 29) (SEQ ID NO.31) (SEQ ID NO:69)(SEQ ID NO:6) A1H5 SEQ ID NO:70 T A GMSVG (SEQ ID NO:10) DIWWD G KK HYNPSLK D D MI F NWYFDV SEQ ID NO:71 SLSSS VGYMH DT FFHR S FQGSGYPFTNO:70 (SEQ ID NO:25) (SEQ ID NO:29) (SEQ ID NO:72) (SEQ ID NO.73) (SEQID NO 6) A4B4(1) SEQ ID NO 48 T A GMSVG (SEQ ID NO:10) DIWWDDKK H YNPSLKD D MI F N F YFDV SEQ ID NO.74 SASSR VGYMH DT LL L D S FQGSGYPFT (SEQ IDNO 19) (SEQ ID NO:20) (SEQ ID NO.39) (SEQ ID NO:75) (SEQ ID NO:6)A4B4L1FR-S28R SEQ ID NO 48 T A GMSVG (SEQ ID NO:10) DIWWDDKK H YNPSLK DD MI F N F YFDV SEQ ID NO 11 SASSR VGYMH DTSKLAS FQGSGYPFT (SEQ IDNO.19) (SEQ ID NO:20) (SEQ ID NO:39) (SEQ ID NO:5) (SEQ ID NO:6)A4B4-F52S SEQ ID NO.48 T A GMSVG (SEQ ID NO:10) DIWWDDKK H YNPSLK D D MIF N F YFDV SEQ ID NO.76 SASSR VGYMH DTS F L D S FQGSGYPFT (SEQ ID NO:19)(SEQ ID NO:20) (SEQ ID NO:39) (SEQ ID NO:77) (SEQ ID NO:6)

[0079] In other embodiments, the antibody is a modified anti-α_(v)β₃antibody, preferably a Vitaxin antibody (see, PCT publications WO98/33919 and WO 00/78815, both by Huse et al., and both of which areincorporated by reference herein in their entireties). Modified IgGs ofthe present invention having longer half-lives than wild type may alsoinclude IgGs whose bioactive sites, such as antigen-binding sites,Fc-receptor binding sites, or complement-binding sites, are modified bygenetic engineering to increase or reduce such activities compared tothe wild type.

[0080] Modification of these and other therapeutic antibodies toincrease the in vivo half-life permits administration of lower effectivedosages and/or less frequent dosing of the therapeutic antibody. Suchmodification to increase in vivo half-life can also be useful to improvediagnostic immunoglobulins as well, for example, permittingadministration of lower doses to achieve sufficient diagnosticsensitivity.

[0081] The present invention also provides fusion proteins comprising abioactive molecule and an hinge-Fc region or a fragment thereof(preferably human) having one or more modifications (i.e.,substitutions, deletions, or insertions) in amino acid residuesidentified to be involved in the interaction between the hinge-Fc regionand the FcRn receptor. In particular, the present invention providesfusion proteins comprising a bioactive molecule recombinantly fused orchemically conjugated (including both covalent and non-covalentconjugations) to a CH2 domain having one or more modifications in aminoacid residues 251-256, 285-290, and/or amino acid residues 308-314,and/or to a CH3 domain having one or more modifications in amino acidresidues 385-389 and/or 428-436, in particular, one or more of the aminoacid substitutions discussed above. The fusion of a bioactive moleculeto a constant domain or a fragment thereof with one or more of suchmodifications increases the in vivo half-life of the bioactive molecule.

[0082] In a preferred embodiment, fusion proteins of the inventioncomprise a bioactive molecule recombinantly fused or chemicallyconjugated to a CH2 domain having one or more amino acid residuesubstitutions in amino acid residues 251-256, 285-290, and/or amino acidresidues 308-314, and/or to a CH3 domain having one or moremodifications in amino acid residues 385-389 and/or 428-436. In certainembodiments, a fusion protein comprises a CH2 domain of IgG molecule inwhich amino acid residues 253, 310, and 313 are not modified. In anotherembodiments, a fusion protein comprises a CH3 domain of IgG molecule inwhich amino acid residues 388, 429, 430, 431, 432, and 435 are notmodified.

[0083] A bioactive molecule can be any polypeptide or synthetic drugknown to one of skill in the art. Preferably, a bioactive molecule is apolypeptide consisting of at least 5, preferably at least 10, at least20, at least 30, at least 40, at least 50, at least 60, at least 70, atleast 80, at least 90 or at least 100 amino acid residues. Examples ofbioactive polypeptides include, but are not limited to, various types ofantibodies, cytokines (e.g., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10,IL-12, IL-15, IFN-γ, IFN-α, and IFN-β), cell adhesion molecules (e.g.,CTLA4, CD2, and CD28), ligands (e.g., TNF-α,, TNF-β, and ananti-angiogenic factor such as endostatin), receptors, antibodies andgrowth factors (e.g., PDGF, EGF, NGF, and KGF).

[0084] A bioactive molecule can also be a therapeutic moiety such as acytotoxin (e.g., a cytostatic or cytocidal agent), a therapeutic agentor a radioactive element (e.g., alpha-emitters, gamma-emitters, etc.).Examples of cytostatic or cytocidal agents include, but are not limitedto, paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin,doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,procaine, tetracaine, lidocaine, propranolol, and puromycin and analogsor homologs thereof. Therapeutic agents include, but are not limited to,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cisdichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

[0085] The present invention also provides polynucleotides comprising anucleotide sequence encoding a modified IgG of the invention andfragments thereof which contain the modified FcRn binding sites withincreased affinity and vectors comprising said polynucleotides.Furthermore, the invention includes polynucleotides that hybridize understringent or lower stringent hybridization conditions to polynucleotidesencoding modified IgGs of the present invention.

[0086] The nucleotide sequence of modified IgGs and the polynucleotidesencoding the same may be obtained by any methods known in the art,including general DNA sequencing method, such as dideoxy chaintermination method (Sanger sequencing), and oligonucleotide priming incombination with PCR, respectively.

5.2. IDENTIFICATION OF MUTATIONS WITHIN THE HINGE-FC REGION OFIMMUNOGLOBULIN MOLECULES

[0087] One or more modifications in amino acid residues 251-256,285-290, 308-314, 385-389, and 428-436 of the constant domain may beintroduced utilizing any technique known to those of skill in the art.The constant domain or fragment thereof having one or more modificationsin amino acid residues 251-256, 285-290, 308-314, 385-389, and 428-436may be screened by, for example, a binding assay to identify theconstant domain or fragment thereof with increased affinity for the FcRnreceptor (e.g., as described in section 5.11, infra). Thosemodifications in the hinge-Fc domain or the fragments thereof whichincrease the affinity of the constant domain or fragment thereof for theFcRn receptor can be introduced into antibodies to increase the in vivohalf-lives of said antibodies. Further, those modifications in theconstant domain or the fragment thereof which increase the affinity ofthe constant domain or fragment thereof for the FcRn can be fused tobioactive molecules to increase the in vivo half-lives of said bioactivemolecules (and, preferably alter (increase or decrease) thebioavailability of the molecule, for example, to increase or decreasetransport to mucosal surfaces (or other target tissue) (e.g., thelungs).

5.2.1. MUTAGENESIS

[0088] Mutagenesis may be performed in accordance with any of thetechniques known in the art including, but not limited to, synthesizingan oligonucleotide having one or more modifications within the sequenceof the constant domain of an antibody or a fragment thereof (e.g., theCH2 or CH3 domain) to be modified. Site-specific mutagenesis allows theproduction of mutants through the use of specific oligonucleotidesequences which encode the DNA sequence of the desired mutation, as wellas a sufficient number of adjacent nucleotides, to provide a primersequence of sufficient size and sequence complexity to form a stableduplex on both sides of the deletion junction being traversed.Typically, a primer of about 17 to about 75 nucleotides or more inlength is preferred, with about 10 to about 25 or more residues on bothsides of the junction of the sequence being altered. A number of suchprimers introducing a variety of different mutations at one or morepositions may be used to generated a library of mutants.

[0089] The technique of site-specific mutagenesis is well known in theart, as exemplified by various publications (see, e.g.,. Kunkel et al.,Methods Enzymol., 154:367-82, 1987, which is hereby incorporated byreference in its entirety). In general, site-directed mutagenesis isperformed by first obtaining a single-stranded vector or melting apartof two strands of a double stranded vector which includes within itssequence a DNA sequence which encodes the desired peptide. Anoligonucleotide primer bearing the desired mutated sequence is prepared,generally synthetically. This primer is then annealed with thesingle-stranded vector, and subjected to DNA polymerizing enzymes suchas T7 DNA polymerase, in order to complete the synthesis of themutation-bearing strand. Thus, a heteroduplex is formed wherein onestrand encodes the original non-mutated sequence and the second strandbears the desired mutation. This heteroduplex vector is then used totransform or transfect appropriate cells, such as E. coli cells, andclones are selected which include recombinant vectors bearing themutated sequence arrangement. As will be appreciated, the techniquetypically employs a phage vector which exists in both a single strandedand double stranded form. Typical vectors useful in site-directedmutagenesis include vectors such as the M13 phage. These phage arereadily commercially available and their use is generally well known tothose skilled in the art. Double stranded plasmids are also routinelyemployed in site directed mutagenesis which eliminates the step oftransferring the gene of interest from a plasmid to a phage.

[0090] Alternatively, the use of PCR™ with commercially availablethermostable enzymes such as Taq DNA polymerase may be used toincorporate a mutagenic oligonucleotide primer into an amplified DNAfragment that can then be cloned into an appropriate cloning orexpression vector. See, e.g., Tomic et al., Nucleic Acids Res.,18(6):1656, 1987, and Upender et al., Biotechniques, 18(1):29-30, 32,1995, for PCR™-mediated mutagenesis procedures, which are herebyincorporated in their entireties. PCR™ employing a thermostable ligasein addition to a thermostable polymerase may also be used to incorporatea phosphorylated mutagenic oligonucleotide into an amplified DNAfragment that may then be cloned into an appropriate cloning orexpression vector (see e.g., Michael, Biotechniques, 16(3):410-2, 1994,which is hereby incorporated by reference in its entirety).

[0091] Other methods known to those of skill in art of producingsequence variants of the Fc domain of an antibody or a fragment thereofcan be used. For example, recombinant vectors encoding the amino acidsequence of the constant domain of an antibody or a fragment thereof maybe treated with mutagenic agents, such as hydroxylamine, to obtainsequence variants.

5.2.2. PANNING

[0092] Vectors, in particular, phage, expressing constant domains orfragments thereof having one or more modifications in amino acidresidues 251-256, 285-290, 308-314, 385-389, and/or 428-436 can bescreened to identify constant domains or fragments thereof havingincreased affinity for FcRn to select out the highest affinity bindersfrom a population of phage. Immunoassays which can be used to analyzebinding of the constant domain or fragment thereof having one or moremodifications in amino acid residues 251-256, 285-290, 308-314, 385-389,and/or 428-436 to the FcRn include, but are not limited to,radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich”immunoassays, and fluorescent immunoassays. Such assays are routine andwell known in the art (see, e.g., Ausubel et al., eds, 1994, CurrentProtocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., NewYork, which is incorporated by reference herein in its entirety).Exemplary immunoassays are described briefly herein below (but are notintended by way of limitation). BIAcore kinetic analysis can also beused to determine the binding on and off rates of a constant domain or afragment thereof having one or more modifications in amino acid residues251-256, 285-290, 308-314, 385-389, and/or 428-436 to the FcRn. BIAcorekinetic analysis comprises analyzing the binding and dissociation of aconstant domain or a fragment thereof having one or more modificationsin amino acid residues 251-256, 285-290, 308-314, 385-389, and/or428-436 from chips with immobilized FcRn on their surface (see section5.1 and the Example section infra).

5.2.3. SEQUENCING

[0093] Any of a variety of sequencing reactions known in the art can beused to directly sequence the nucleotide sequence encoding constantdomains or fragments thereof having one or more modifications in aminoacid residues 251-256, 285-290, 308-314, 385-389, and/or 428-436.Examples of sequencing reactions include those based on techniquesdeveloped by Maxim and Gilbert (Proc. Natl. Acad. Sci. USA, 74:560,1977) or Sanger (Proc. Natl. Acad. Sci. USA, 74:5463, 1977). It is alsocontemplated that any of a variety of automated sequencing procedurescan be utilized (Bio/Techniques, 19:448, 1995), including sequencing bymass spectrometry (see, e.g., PCT Publication No. WO 94/16101, Cohen etal., Adv. Chromatogr., 36:127-162, 1996, and Griffin et al., Appl.Biochem. Biotechnol., 38:147-159, 1993).

5.3. RECOMBINANT METHODS OF PRODUCING ANTIBODIES

[0094] The antibodies of the invention or fragments thereof can beproduced by any method known in the art for the synthesis of antibodies,in particular, by chemical synthesis or preferably, by recombinantexpression techniques.

[0095] The nucleotide sequence encoding an antibody may be obtained fromany information available to those of skill in the art (i.e., fromGenbank, the literature, or by routine cloning). If a clone containing anucleic acid encoding a particular antibody or an epitope-bindingfragment thereof is not available, but the sequence of the antibodymolecule or epitope-binding fragment thereof is known, a nucleic acidencoding the immunoglobulin may be chemically synthesized or obtainedfrom a suitable source (e.g., an antibody cDNA library, or a cDNAlibrary generated from, or nucleic acid, preferably poly A⁺ RNA,isolated from any tissue or cells expressing the antibody, such ashybridoma cells selected to express an antibody) by PCR amplificationusing synthetic primers hybridizable to the 3′ and 5′ ends of thesequence or by cloning using an oligonucleotide probe specific for theparticular gene sequence to identify, e.g., a cDNA clone from a cDNAlibrary that encodes the antibody. Amplified nucleic acids generated byPCR may then be cloned into replicable cloning vectors using any methodwell known in the art.

[0096] Once the nucleotide sequence of the antibody is determined, thenucleotide sequence of the antibody may be manipulated using methodswell known in the art for the manipulation of nucleotide sequences,e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc.(see, for example, the techniques described in Sambrook et al., 1990,Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.; and Ausubel et al., eds., 1998,Current Protocols in Molecular Biology, John Wiley & Sons, NY, which areboth incorporated by reference herein in their entireties), to generateantibodies having a different amino acid sequence by, for example,introducing amino acid substitutions, deletions, and/or insertions intothe epitope-binding domain regions of the antibodies and preferably,into the hinge-Fc regions of the antibodies which are involved in theinteraction with the FcRn. In a preferred embodiment, antibodies havingone or more modifications in amino acid residues 251-256, 285-290,308-314, 385-389, and 428-436 are generated.

[0097] Recombinant expression of an antibody requires construction of anexpression vector containing a nucleotide sequence that encodes theantibody. Once a nucleotide sequence encoding an antibody molecule or aheavy or light chain of an antibody, or portion thereof (preferably, butnot necessarily, containing the heavy or light chain variable region)has been obtained, the vector for the production of the antibodymolecule may be produced by recombinant DNA technology using techniqueswell known in the art. Thus, methods for preparing a protein byexpressing a polynucleotide containing an antibody encoding nucleotidesequence are described herein. Methods which are well known to thoseskilled in the art can be used to construct expression vectorscontaining antibody coding sequences and appropriate transcriptional andtranslational control signals. These methods include, for example, invitro recombinant DNA techniques, synthetic techniques, and in vivogenetic recombination. The invention, thus, provides replicable vectorscomprising a nucleotide sequence encoding the constant region of theantibody molecule with one or more modifications in the amino acidresidues involved in the interaction with the FcRn (see, e.g., PCTPublication WO 86/05807; PCT Publication WO 89/01036; and U.S. Pat. No.5,122,464). The nucleotide sequence encoding the heavy-chain variableregion, light-chain variable region, both the heavy-chain andlight-chain variable regions, an epitope-binding fragment of the heavy-and/or light-chain variable region, or one or more complementaritydetermining regions (CDRs) of an antibody may be cloned into such avector for expression.

[0098] The expression vector is transferred to a host cell byconventional techniques and the transfected cells are then cultured byconventional techniques to produce an antibody having an increasedaffinity for the FcRn and an increased in vivo half-life. Thus, theinvention includes host cells containing a polynucleotide encoding anantibody, a constant domain or a FcRn binding fragment thereof havingone or more modifications in amino acid residues 251-256, 285-290,308-314, 385-389, and/or 428-436, preferably, operably linked to aheterologous promoter.

[0099] A variety of host-expression vector systems may be utilized toexpress the antibody molecules of the invention. Such host-expressionsystems represent vehicles by which the coding sequences of interest maybe produced and subsequently purified, but also represent cells whichmay, when transformed or transfected with the appropriate nucleotidecoding sequences, express an antibody molecule of the invention in situ.These include, but are not limited to, microorganisms such as bacteria(e.g., E. coli and B. subtilis) transformed with recombinantbacteriophage DNA, plasmid DNA or cosmid DNA expression vectorscontaining antibody coding sequences; yeast (e.g., Saccharomyces andPichia) transformed with recombinant yeast expression vectors containingantibody coding sequences; insect cell systems infected with recombinantvirus expression vectors (e.g., baculovirus) containing antibody codingsequences; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; and tobacco mosaic virus,TMV) or transformed with recombinant plasmid expression vectors (e.g.,Ti plasmid) containing antibody coding sequences; and mammalian cellsystems (e.g., COS, CHO, BHK, 293, 3T3 and NSO cells) harboringrecombinant expression constructs containing promoters derived from thegenome of mammalian cells (e.g., metallothionein promoter) or frommammalian viruses (e.g., the adenovirus late promoter; the vacciniavirus 7.5K promoter). Preferably, bacterial cells such as Escherichiacoli, and more preferably, eukaryotic cells, especially for theexpression of whole recombinant antibody molecule, are used for theexpression of a recombinant antibody molecule. For example, mammaliancells such as Chinese hamster ovary cells (CHO), in conjunction with avector such as the major intermediate early gene promoter element fromhuman cytomegalovirus is an effective expression system for antibodies(Foecking et al., Gene, 45:101, 1986, and Cockett et al.,Bio/Technology, 8:2, 1990).

[0100] In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of pharmaceuticalcompositions of an antibody molecule, vectors which direct theexpression of high levels of fusion protein products that are readilypurified may be desirable. Such vectors include, but are not limited to,the E. coli expression vector pUR278 (Ruther et al., EMBO, 12:1791,1983), in which the antibody coding sequence may be ligated individuallyinto the vector in frame with the lacZ coding region so that a fusionprotein is produced; and pIN vectors (Inouye & Inouye, Nucleic AcidsRes., 13:3101-3109, 1985 and Van Heeke & Schuster, J. Biol. Chem.,24:5503-5509, 1989).

[0101] In an insect system, Autographa californica nuclear polyhedrosisvirus (AcNPV) is used as a vector to express foreign genes. The virusgrows in Spodoptera frugiperda cells. The antibody coding sequence maybe cloned individually into non-essential regions (for example thepolyhedrin gene) of the virus and placed under control of an AcNPVpromoter (for example the polyhedrin promoter).

[0102] In mammalian host cells, a number of viral-based expressionsystems may be utilized to express an antibody molecule of theinvention. In cases where an adenovirus is used as an expression vector,the antibody coding sequence of interest may be ligated to an adenovirustranscription/translation control complex, e.g., the late promoter andtripartite leader sequence. This chimeric gene may then be inserted inthe adenovirus genome by in vitro or in vivo recombination. Insertion ina non-essential region of the viral genome (e.g., region E1 or E3) willresult in a recombinant virus that is viable and capable of expressingthe antibody molecule in infected hosts (e.g., see Logan & Shenk, Proc.Natl. Acad. Sci. USA, 81:355-359, 1984). Specific initiation signals mayalso be required for efficient translation of inserted antibody codingsequences. These signals include the ATG initiation codon and adjacentsequences. Furthermore, the initiation codon must be in phase with thereading frame of the desired coding sequence to ensure translation ofthe entire insert. These exogenous translational control signals andinitiation codons can be of a variety of origins, both natural andsynthetic. The efficiency of expression may be enhanced by the inclusionof appropriate transcription enhancer elements, transcriptionterminators, etc. (see, e.g., Bitter et al., Methods in Enzymol.,153:516-544, 1987).

[0103] In addition, a host cell strain may be chosen which modulates theexpression of the antibody sequences, or modifies and processes theantibody in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the antibody. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the antibody expressed. To this end,eukaryotic host cells which possess the cellular machinery for properprocessing of the primary transcript, glycosylation, and phosphorylationof the gene product may be used. Such mammalian host cells include butare not limited to CHO, VERY, BHK, HeLa, COS, MDCK, 293, 3T3, W138, andin particular, myeloma cells such as NS0 cells, and related cell lines,see, for example, Morrison et al., U.S. Pat. No. 5,807,715, which ishereby incorporated by reference in its entirety.

[0104] For long-term, high-yield production of recombinant antibodies,stable expression is preferred. For example, cell lines which stablyexpress the antibody molecule may be engineered. Rather than usingexpression vectors which contain viral origins of replication, hostcells can be transformed with DNA controlled by appropriate expressioncontrol elements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which express the antibodymolecule. Such engineered cell lines may be particularly useful inscreening and evaluation of compositions that interact directly orindirectly with the antibody molecule.

[0105] A number of selection systems may be used, including but notlimited to, the herpes simplex virus thymidine kinase (Wigler et al.,Cell, 11:223, 1977), hypoxanthine-guanine phosphoribosyltransferase(Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA, 48:202, 1992), andadenine phosphoribosyltransferase (Lowy et al., Cell, 22:8-17, 1980)genes can be employed in tk⁻, hgprt⁻ or aprt⁻ cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., Natl. Acad. Sci. USA, 77:357, 1980 and O'Hare et al., Proc.Natl. Acad. Sci. USA, 78:1527, 1981); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA, 78:2072,1981); neo, which confers resistance to the aminoglycoside G-418 (Wu andWu, Biotherapy, 3:87-95, 1991; Tolstoshev, Ann. Rev. Pharmacol.Toxicol., 32:573-596, 1993; Mulligan, Science, 260:926-932, 1993; andMorgan and Anderson, Ann. Rev. Biochem., 62: 191-217, 1993; and May, TIBTECH, 11(5):155-2 15, 1993); and hygro, which confers resistance tohygromycin (Santerre et al., Gene, 30:147, 1984). Methods commonly knownin the art of recombinant DNA technology may be routinely applied toselect the desired recombinant clone, and such methods are described,for example, in Ausubel et al. (eds.), 1993, Current Protocols inMolecular Biology, John Wiley & Sons, NY; Kriegler, 1990, Gene Transferand Expression, A Laboratory Manual, Stockton Press, NY; in Chapters 12and 13, Dracopoli et al. (eds), 1994, Current Protocols in HumanGenetics, John Wiley & Sons, NY; and Colberre-Garapin et al., J. Mol.Biol., 150:1, 1981, which are incorporated by reference herein in theirentireties.

[0106] The expression levels of an antibody molecule can be increased byvector amplification (for a review, see Bebbington and Hentschel, 1987,The use of vectors based on gene amplification for the expression ofcloned genes in mammalian cells in DNA cloning, Vol.3. Academic Press,New York). When a marker in the vector system expressing antibody isamplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the antibody gene, production ofthe antibody will also increase (Crouse et al., Mol., Cell. Biol.,3:257, 1983).

[0107] The host cell may be co-transfected with two expression vectorsof the invention, the first vector encoding a heavy chain derivedpolypeptide and the second vector encoding a light chain derivedpolypeptide. The two vectors may contain identical selectable markerswhich enable equal expression of heavy and light chain polypeptides ordifferent selectable markers to ensure maintenance of both plasmids.Alternatively, a single vector may be used which encodes, and is capableof expressing, both heavy and light chain polypeptides. In suchsituations, the light chain should be placed before the heavy chain toavoid an excess of toxic free heavy chain (Proudfoot, Nature, 322:52,1986; and Kohler, Proc. Natl. Acad. Sci. USA, 77:2 197, 1980). Thecoding sequences for the heavy and light chains may comprise cDNA orgenomic DNA.

[0108] Once an antibody molecule of the invention has been produced byrecombinant expression, it may be purified by any method known in theart for purification of an immunoglobulin molecule, for example, bychromatography (e.g., ion exchange, affinity, particularly by affinityfor the specific antigen after Protein A purification, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard techniques for the purification of proteins. Further, theantibodies of the present invention or fragments thereof may be fused toheterologous polypeptide sequences described herein or otherwise knownin the art to facilitate purification.

5.3.1. ANTIBODY CONJUGATES

[0109] The present invention encompasses antibodies recombinantly fusedor chemically conjugated (including both covalently and non-covalentlyconjugations) to heterologous polypeptides (i.e., an unrelatedpolypeptide; or portion thereof, preferably at least 10, at least 20, atleast 30, at least 40, at least 50, at least 60, at least 70, at least80, at least 90 or at least 100 amino acids of the polypeptide) togenerate fusion proteins. The fusion does not necessarily need to bedirect, but may occur through linker sequences. Antibodies fused orconjugated to heterologous polypeptides may also be used in in vitroimmunoassays and purification methods using methods known in the art.See e.g., PCT Publication No. WO 93/21232; EP 439,095; Naramura et al.,Immunol. Lett., 39:91-99, 1994; U.S. Pat. No. 5,474,981; Gillies et al.,PNAS, 89:1428-1432, 1992; and Fell et al., J. Immunol., 146:2446-2452,1991, which are incorporated herein by reference in their entireties.

[0110] Antibodies can be fused to marker sequences, such as a peptide tofacilitate purification. In preferred embodiments, the marker amino acidsequence is a hexa-histidine peptide, such as the tag provided in a pQEvector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311),among others, many of which are commercially available. As described inGentz et al., Proc. Natl. Acad. Sci. USA, 86:821-824, 1989, forinstance, hexa-histidine provides for convenient purification of thefusion protein. Other peptide tags useful for purification include, butare not limited to, the hemagglutinin “HA” tag, which corresponds to anepitope derived from the influenza hemagglutinin protein (Wilson et al.,Cell, 37:767 1984) and the “flag” tag (Knappik et al., Biotechniques,17(4):754-761, 1994).

[0111] The present invention also encompasses antibodies conjugated to adiagnostic or therapeutic agent or any other molecule for which in vivohalf-life is desired to be increased. The antibodies can be useddiagnostically to, for example, monitor the development or progressionof a disease, disorder or infection as part of a clinical testingprocedure to, e.g., determine the efficacy of a given treatment regimen.Detection can be facilitated by coupling the antibody to a detectablesubstance. Examples of detectable substances include various enzymes,prosthetic groups, fluorescent materials, luminescent materials,bioluminescent materials, radioactive materials, positron emittingmetals, and nonradioactive paramagnetic metal ions. The detectablesubstance may be coupled or conjugated either directly to the antibodyor indirectly, through an intermediate (such as, for example, a linkerknown in the art) using techniques known in the art. See, for example,U.S. Pat. No. 4,741,900 for metal ions which can be conjugated toantibodies for use as diagnostics according to the present invention.Examples of suitable enzymes include horseradish peroxidase, alkalinephosphatase, β-galactosidase, or acetylcholinesterase; examples ofsuitable prosthetic group complexes include streptavidin/biotin andavidinibiotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin;and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ¹¹¹Inor ^(99m)Tc.

[0112] An antibody may be conjugated to a therapeutic moiety such as acytotoxin (e.g., a cytostatic or cytocidal agent), a therapeutic agentor a radioactive element (e.g., alpha-emitters, gamma-emitters, etc.).Cytotoxins or cytotoxic agents include any agent that is detrimental tocells. Examples include paclitaxol, cytochalasin B, gramicidin D,ethidium bromide, emetine, mitomycin, etoposide, tenoposide,vincristine, vinblastine, colchicin, doxorubicin, daunorubicin,dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D,1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,propranolol, and puromycin and analogs or homologs thereof. Therapeuticagents include, but are not limited to, antimetabolites (e.g.,methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,5-fluorouracil decarbazine), alkylating agents (e.g, mechlorethamine,thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cisdichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines(e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics(e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, andanthramycin (AMC)), and anti-mitotic agents (e.g., vincristine andvinblastine).

[0113] Further, an antibody may be conjugated to a therapeutic agent ordrug moiety that modifies a given biological response. Therapeuticagents or drug moieties are not to be construed as limited to classicalchemical therapeutic agents. For example, the drug moiety may be aprotein or polypeptide possessing a desired biological activity. Suchproteins may include, for example, a toxin such as abrin, ricin A,pseudomonas exotoxin, or diphtheria toxin; a protein such as tumornecrosis factor, α-interferon (IFN-α), β-interferon (IFN-β), nervegrowth factor (NGF), platelet derived growth factor (PDGF), tissueplasminogen activator (TPA), an apoptotic agent (e.g., TNF-α, TNF-β, AIMI as disclosed in PCT Publication No. WO 97/33899), AIM II (see, PCTPublication No. WO 97/34911), Fas Ligand (Takahashi et al., J. Immunol.,6:1567-1574, 1994), and VEGI (PCT Publication No. WO 99/23105), athrombotic agent or an anti-angiogenic agent (e.g., angiostatin orendostatin); or a biological response modifier such as, for example, alymphokine (e.g., interleukin-1 (“IL-1”), interleukin-2 (“IL-2”),interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor(“GM-CSF”), and granulocyte colony stimulating factor (“G-CSF”)), or agrowth factor (e.g., growth hormone (“GH”)).

[0114] Techniques for conjugating such therapeutic moieties toantibodies are well known; see, e.g., Arnon et al., “MonoclonalAntibodies For Immunotargeting Of Drugs In Cancer Therapy”, inMonoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), 1985,pp. 243-56, Alan R. Liss, Inc.); Hellstrom et al., “Antibodies For DrugDelivery”, in Controlled Drug Delivery (2nd Ed.), Robinson et al.(eds.), 1987, pp. 623-53, Marcel Dekker, Inc. ); Thorpe, “AntibodyCarriers Of Cytotoxic Agents In Cancer Therapy: A Review”, in MonoclonalAntibodies '84:Biological And Clinical Applications, Pinchera et al.(eds.), 1985, pp. 475-506); “Analysis, Results, And Future ProspectiveOf The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.),1985, pp. 303-16, Academic Press; and Thorpe et al., Immunol.Recombinant expression vector., 62:119-58, 1982.

[0115] An antibody or fragment thereof, with or without a therapeuticmoiety conjugated to it, administered alone or in combination withcytotoxic factor(s) and/or cytokine(s) can be used as a therapeutic.

[0116] Alternatively, an antibody can be conjugated to a second antibodyto form an antibody heteroconjugate as described by Segal in U.S. Pat.No. 4,676,980, which is incorporated herein by reference in itsentirety.

[0117] Antibodies may also be attached to solid supports, which areparticularly useful for immunoassays or purification of the targetantigen. Such solid supports include, but are not limited to, glass,cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride orpolypropylene.

5.4 METHODS OF PRODUCING FUSION PROTEINS

[0118] Fusion proteins can be produced by standard recombinant DNAtechniques or by protein synthetic techniques, e.g., by use of a peptidesynthesizer. For example, a nucleic acid molecule encoding a fusionprotein can be synthesized by conventional techniques includingautomated DNA synthesizers. Alternatively, PCR amplification of genefragments can be carried out using anchor primers which give rise tocomplementary overhangs between two consecutive gene fragments which cansubsequently be annealed and reamplified to generate a chimeric genesequence (see, e.g., Current Protocols in Molecular Biology, Ausubel etal., eds., John Wiley & Sons, 1992). Moreover, a nucleic acid encoding abioactive molecule can be cloned into an expression vector containingthe Fc domain or a fragment thereof such that the bioactive molecule islinked in-frame to the constant domain or fragment thereof.

[0119] Methods for fusing or conjugating polypeptides to the constantregions of antibodies are known in the art. See, e.g., U.S. Pat. Nos.5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, 5,723,125,5,783,181, 5,908,626, 5,844,095, and 5,112,946; EP 307,434; EP 367,166;EP 394,827; PCT publications WO 91/06570, WO 96/04388, WO 96/22024, WO97/34631, and WO 99/04813; Ashkenazi et al., Proc. Natl. Acad. Sci. USA,88: 10535-10539, 1991; Traunecker et al., Nature, 331:84-86, 1988; Zhenget al., J. Immunol., 154:5590-5600, 1995; and Vil et al., Proc. Natl.Acad. Sci. USA, 89:11337-11341, 1992, which are incorporated herein byreference in their entireties.

[0120] The nucleotide sequence encoding a bioactive molecule may beobtained from any information available to those of skill in the art(e.g., from Genbank, the literature, or by routine cloning), and thenucleotide sequence encoding a constant domain or a fragment thereofwith increased affinity for the FcRn may be determined by sequenceanalysis of mutants produced using techniques described herein, or maybe obtained from Genbank or the literature. The nucleotide sequencecoding for a fusion protein can be inserted into an appropriateexpression vector, i.e., a vector which contains the necessary elementsfor the transcription and translation of the inserted protein-codingsequence. A variety of host-vector systems may be utilized in thepresent invention to express the protein-coding sequence. These includebut are not limited to mammalian cell systems infected with virus (e.g.,vaccinia virus, adenovirus, etc.); insect cell systems infected withvirus (e.g., baculovirus); microorganisms such as yeast containing yeastvectors; or bacteria transformed with bacteriophage, DNA, plasmid DNA,or cosmid DNA. The expression elements of vectors vary in theirstrengths and specificities. Depending on the host-vector systemutilized, any one of a number of suitable transcription and translationelements may be used.

[0121] The expression of a fusion protein may be controlled by anypromoter or enhancer element known in the art. Promoters which may beused to control the expression of the gene encoding fusion proteininclude, but are not limited to, the SV40 early promoter region(Bernoist and Chambon, Nature, 290:304-310, 1981), the promotercontained in the 3′ long terminal repeat of Rous sarcoma virus(Yamamoto, et al., Cell, 22:787-797, 1980), the herpes thymidine kinasepromoter (Wagner et al., Proc. Natl. Acad. Sci. U.S.A., 78:1441-1445,1981), the regulatory sequences of the metallothionein gene (Brinster etal., Nature, 296:39-42, 1982), the tetracycline (Tet) promoter (Gossenet al., Proc. Nat. Acad. Sci. USA, 89:5547-5551, 1995); prokaryoticexpression vectors such as the β-lactamase promoter (Villa-Kamaroff, etal., Proc. Natl. Acad. Sci. U.S.A., 75:3727-3731, 1978), or the tacpromoter (DeBoer, et al., Proc. Natl. Acad. Sci. U.S.A., 80:21-25, 1983;see also “Useful proteins from recombinant bacteria” in ScientificAmerican, 242:74-94, 1980); plant expression vectors comprising thenopaline synthetase promoter region (Herrera-Estrella et al., Nature,303:209-213, 1983) or the cauliflower mosaic virus 35S RNA promoter(Gardner, et al., Nucl. Acids Res., 9:2871, 1981), and the promoter ofthe photosynthetic enzyme ribulose biphosphate carboxylase(Herrera-Estrella et al., Nature, 310:115-120, 1984); promoter elementsfrom yeast or other fungi such as the Gal 4 promoter, the ADC (alcoholdehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkalinephosphatase promoter, and the following animal transcriptional controlregions, which exhibit tissue specificity and have been utilized intransgenic animals: elastase I gene control region which is active inpancreatic acinar cells (Swift et al., Cell 38:639-646, 1984; Ornitz etal., 50:399-409, Cold Spring Harbor Symp. Quant. Biol., 1986; MacDonald,Hepatology 7:425-515, 1987); insulin gene control region which is activein pancreatic beta cells (Hanahan, Nature 315:115-122, 1985),immunoglobulin gene control region which is active in lymphoid cells(Grosschedl et al., Cell, 38:647-658, 1984; Adames et al., Nature318:533-538, 1985; Alexander et al., Mol. Cell. Biol., 7:1436-1444,1987), mouse mammary tumor virus control region which is active intesticular, breast, lymphoid and mast cells (Leder et al., Cell,45:485-495, 1986), albumin gene control region which is active in liver(Pinkert et al., Genes and Devel., 1:268-276, 1987), α-fetoprotein genecontrol region which is active in liver (Krumlauf et al., Mol. Cell.Biol., 5:1639-1648, 1985; Hammer et al., Science, 235:53-58, 1987; α1-antitrypsin gene control region which is active in the liver (Kelseyet al., Genes and Devel., 1:161-171, 1987), beta-globin gene controlregion which is active in myeloid cells (Mogram et al., Nature,315:338-340, 1985; Kollias et al., Cell, 46:89-94, 1986; myelin basicprotein gene control region which is active in oligodendrocyte cells inthe brain (Readhead et al., Cell, 48:703-712, 1987); myosin lightchain-2 gene control region which is active in skeletal muscle (Sani,Nature, 314:283-286, 1985); neuronal-specific enolase (NSE) which isactive in neuronal cells (Morelli et al., Gen. Virol., 80:571-83, 1999);brain-derived neurotrophic factor (BDNF) gene control region which isactive in neuronal cells (Tabuchi et al., Biochem. Biophysic. Res.Comprising., 253:818-823, 1998); glial fibrillary acidic protein (GFAP)promoter which is active in astrocytes (Gomes et al., Braz. J. Med.Biol. Res., 32(5):619-631, 1999; Morelli et al., Gen. Virol., 80:571-83,1999) and gonadotropic releasing hormone gene control region which isactive in the hypothalamus (Mason et al., Science, 234:1372-1378, 1986).

[0122] In a specific embodiment, the expression of a fusion protein isregulated by a constitutive promoter. In another embodiment, theexpression of a fusion protein is regulated by an inducible promoter. Inaccordance with these embodiments, the promoter may be a tissue-specificpromoter.

[0123] In a specific embodiment, a vector is used that comprises apromoter operably linked to a fusion protein-encoding nucleic acid, oneor more origins of replication, and, optionally, one or more selectablemarkers (e.g., an antibiotic resistance gene).

[0124] In mammalian host cells, a number of viral-based expressionsystems may be utilized. In cases where an adenovirus is used as anexpression vector, the fusion protein coding sequence may be ligated toan adenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene may then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) will result in a recombinant virus that is viable and capable ofexpressing the antibody molecule in infected hosts (e.g., see Logan &Shenk, Proc. Natl. Acad. Sci. USA, 81:355-359, 1984). Specificinitiation signals may also be required for efficient translation ofinserted fusion protein coding sequences. These signals include the ATGinitiation codon and adjacent sequences. Furthermore, the initiationcodon must be in phase with the reading frame of the desired codingsequence to ensure translation of the entire insert. These exogeneoustranslation control signals and initiation codons can be of a variety oforigins, both natural and synthetic. The efficiency of expression may beenhanced by the inclusion of appropriate transcription enhancerelements, transcription terminators, etc. (see Bitter et al., Methods inEnzymol., 153:516-544, 1987).

[0125] Expression vectors containing inserts of a gene encoding a fusionprotein can be identified by three general approaches: (a) nucleic acidhybridization, (b) presence or absence of “marker” gene functions, and(c) expression of inserted sequences. In the first approach, thepresence of a gene encoding a fusion protein in an expression vector canbe detected by nucleic acid hybridization using probes comprisingsequences that are homologous to an inserted gene encoding the fusionprotein. In the second approach, the recombinant vector/host system canbe identified and selected based upon the presence or absence of certain“marker” gene functions (e.g. thymidine kinase activity, resistance toantibiotics, transformation phenotype, occlusion body formation inbaculovirus, etc.) caused by the insertion of a nucleotide sequenceencoding a fusion protein in the vector. For example, if the nucleotidesequence encoding the fusion protein is inserted within the marker genesequence of the vector, recombinants containing the gene encoding thefusion protein insert can be identified by the absence of the markergene function. In the third approach, recombinant expression vectors canbe identified by assaying the gene product (i.e., fusion protein)expressed by the recombinant. Such assays can be based, for example, onthe physical or functional properties of the fusion protein in in vitroassay systems, e.g., binding with anti-bioactive molecule antibody.

[0126] In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Expression from certainpromoters can be elevated in the presence of certain inducers; thus,expression of the genetically engineered fusion protein may becontrolled. Furthermore, different host cells have characteristic andspecific mechanisms for the translational and post-translationalprocessing and modification (e.g., glycosylation, phosphorylation ofproteins). Appropriate cell lines or host systems can be chosen toensure the desired modification and processing of the foreign proteinexpressed. For example, expression in a bacterial system will produce anunglycosylated product and expression in yeast will produce aglycosylated product. Eukaryotic host cells which possess the cellularmachinery for proper processing of the primary transcript,glycosylation, and phosphorylation of the gene product may be used. Suchmammalian host cells include but are not limited to CHO, VERY, BHK,HeLa, COS, MDCK, 293, 3T3, WI38, and in particular, neuronal cell linessuch as, for example, SK-N-AS, SK-N-FJ, SK-N-DZ human neuroblastomas(Sugimoto et al., J. Natl. Cancer Inst., 73: 51-57, 1984), SK-N-SH humanneuroblastoma (Biochim. Biophys. Acta, 704: 450-460, 1982), Daoy humancerebellar medulloblastoma (He et al., Cancer Res., 52: 1144-1148, 1992)DBTRG-05MG glioblastoma cells (Kruse et al., 1992, In Vitro Cell. Dev.Biol., 28A:609-614, 1992), IMR-32 human neuroblastoma (Cancer Res., 30:

[0127]2110-2118, 1970), 1321N1 human astrocytoma (Proc. Natl Acad. Sci.USA, 74: 4816, 1997), MOG-G-CCM human astrocytoma (Br. J. Cancer, 49:269, 1984), U87MG human glioblastoma-astrocytoma (Acta Pathol.Microbiol. Scand., 74: 465-486, 1968), A172 human glioblastoma (Olopadeet al., Cancer Res., 52: 2523-2529, 1992), C6 rat glioma cells (Benda etal., Science, 161: 370-371, 1968), Neuro-2a mouse neuroblastoma (Proc.Natl. Acad. Sci. USA, 65: 129-136, 1970), NB41A3 mouse neuroblastoma(Proc. Natl. Acad. Sci. USA, 48: 1184-1190, 1962), SCP sheep choroidplexus (Bolin et al., J. Virol. Methods, 48: 211-221, 1994), G355-5,PG-4 Cat normal astrocyte (Haapala et al., J. Virol., 53: 827-833,1985), Mpf ferret brain (Trowbridge et al., In Vitro, 18: 952-960,1982), and normal cell lines such as, for example, CTX TNA2 rat normalcortex brain (Radany et al., Proc. Natl. Acad. Sci. USA, 89: 6467-6471,1992) such as, for example, CRL7030 and Hs578Bst. Furthermore, differentvector/host expression systems may effect processing reactions todifferent degrees.

[0128] For long-term, high-yield production of recombinant proteins,stable expression is preferred. For example, cell lines which stablyexpress the fusion protein may be engineered. Rather than usingexpression vectors which contain viral origins of replication, hostcells can be transformed with DNA controlled by appropriate expressioncontrol elements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched medium, and then areswitched to a selective medium. The selectable marker in the recombinantplasmid confers resistance to the selection and allows cells to stablyintegrate the plasmid into their chromosomes and grow to form foci whichin turn can be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines that express thedifferentially expressed or pathway gene protein. Such engineered celllines may be particularly useful in screening and evaluation ofcompounds that affect the endogenous activity of the differentiallyexpressed or pathway gene protein.

[0129] A number of selection systems may be used, including but notlimited to the herpes simplex virus thymidine kinase (Wigler, et al.,Cell, 11:223, 1997), hypoxanthine-guanine phosphoribosyltransferase(Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA, 48:2026, 1962), andadenine phosphoribosyltransferase (Lowy, et al., 1980, Cell, 22:817,1980) genes can be employed in tk−, hgprt− or aprt− cells, respectively.Also, antimetabolite resistance can be used as the basis of selectionfor dhfr, which confers resistance to methotrexate (Wigler, et al.,Natl. Acad. Sci. USA, 77:3567, 1980; O'Hare, et al., Proc. Natl. Acad.Sci. USA, 78:1527, 1981); gpt, which confers resistance to mycophenolicacid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA, 78:2072, 1981); neo,which confers resistance to the aminoglycoside G-418 (Colberre-Garapin,et al., J. Mol. Biol., 150:1, 1981); and hygro, which confers resistanceto hygromycin (Santerre, et al., Gene, 30:147, 1984) genes.

[0130] Once a fusion protein of the invention has been produced byrecombinant expression, it may be purified by any method known in theart for purification of a protein, for example, by chromatography (e.g.,ion exchange, affinity, particularly by affinity for the specificantigen after Protein A, and sizing column chromatography),centrifugation, differential solubility, or by any other standardtechnique for the purification of proteins.

5.5. PROPHYLACTIC AND THERAPEUTIC USES OF ANTIBODIES

[0131] The present invention encompasses antibody-based therapies whichinvolve administering antibodies to an animal, preferably a mammal, andmost preferably a human, for preventing, treating, or amelioratingsymptoms associated with a disease, disorder, or infection. Prophylacticand therapeutic compounds of the invention include, but are not limitedto, antibodies and nucleic acids encoding antibodies. Antibodies may beprovided in pharmaceutically acceptable compositions as known in the artor as described herein.

[0132] Antibodies of the present invention that function as antagonistsof a disease, disorder, or infection can be administered to an animal,preferably a mammal and most preferably a human, to treat, prevent orameliorate one or more symptoms associated with the disease, disorder,or infection. For example, antibodies which disrupt or prevent theinteraction between a viral antigen and its host cell receptor may beadministered to an animal, preferably a mammal and most preferably ahuman, to treat, prevent or ameliorate one or more symptoms associatedwith a viral infection.

[0133] In a specific embodiment, an antibody or fragment thereofprevents a viral or bacterial antigen from binding to its host cellreceptor by at least 99%, at least 95%, at least 90%, at least 85%, atleast 80%, at least 75%, at least 70%, at least 60%, at least 50%, atleast 45%, at least 40%, at least 45%, at least 35%, at least 30%, atleast 25%, at least 20%, or at least 10% relative to antigen binding toits host cell receptor in the absence of said antibodies. In anotherembodiment, a combination of antibodies prevent a viral or bacterialantigen from binding to its host cell receptor by at least 99%, at least95%, at least 90%, at least 85%, at least 80%, at least 75%, at least70%, at least 60%, at least 50%, at least 45%, at least 40%, at least45%, at least 35%, at least 30%, at least 25%, at least 20%, or at least10% relative to antigen binding to its host cell receptor in the absenceof said antibodies. In a preferred embodiment, the antibody is used totreat or prevent RSV infection

[0134] Antibodies which do not prevent a viral or bacterial antigen frombinding its host cell receptor but inhibit or downregulate viral orbacterial replication can also be administered to an animal to treat,prevent or ameliorate one or more symptoms associated with a viral orbacterial infection. The ability of an antibody to inhibit ordownregulate viral or bacterial replication may be determined bytechniques described herein or otherwise known in the art. For example,the inhibition or downregulation of viral replication can be determinedby detecting the viral titer in the animal.

[0135] In a specific embodiment, an antibody inhibits or downregulatesviral or bacterial replication by at least 99%, at least 95%, at least90%, at least 85%, at least 80%, at least 75%, at least 70%, at least60%, at least 50%, at least 45%, at least 40%, at least 45%, at least35%, at least 30%, at least 25%, at least 20%, or at least 10% relativeto viral or bacterial replication in absence of said antibody. Inanother embodiment, a combination of antibodies inhibit or downregulateviral or bacterial replication by at least 99%, at least 95%, at least90%, at least 85%, at least 80%, at least 75%, at least 70%, at least60%, at least 50%, at least 45%, at least 40%, at least 45%, at least35%, at least 30%, at least 25%, at least 20%, or at least 10% relativeto viral or bacterial replication in absence of said antibodies.

[0136] Antibodies can also be used to prevent, inhibit or reduce thegrowth or metastasis of cancerous cells. In a specific embodiment, anantibody inhibits or reduces the growth or metastasis of cancerous cellsby at least 99%, at least 95%, at least 90%, at least 85%, at least 80%,at least 75%, at least 70%, at least 60%, at least 50%, at least 45%, atleast 40%, at least 45%, at least 35%, at least 30%, at least 25%, atleast 20%, or at least 10% relative to the growth or metastasis inabsence of said antibody. In another embodiment, a combination ofantibodies inhibits or reduces the growth or metastasis of cancer by atleast 99%, at least 95%, at least 90%, at least 85%, at least 80%, atleast 75%, at least 70%, at least 60%, at least 50%, at least 45%, atleast 40%, at least 45%, at least 35%, at least 30%, at least 25%, atleast 20%, or at least 10% relative to the growth or metastasis inabsence of said antibodies. Examples of cancers include, but are notlimited to, leukemia (e.g, acute leukemia such as acute lymphocyticleukemia and acute myelocytic leukemia), neoplasms, tumors (e.g.,fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, andretinoblastoma), heavy chain disease, metastases, or any disease ordisorder characterized by uncontrolled cell growth.

[0137] Antibodies can also be used to reduce the inflammationexperienced by animals, particularly mammals, with inflammatorydisorders. In a specific embodiment, an antibody reduces theinflammation in an animal by at least 99%, at least 95%, at least 90%,at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, atleast 50%, at least 45% at least 40%, at least 45%, at least 35%, atleast 30%, at least 25%, at least 20%, or at least 10% relative to theinflammation in an animal in the not administered said antibody. Inanother embodiment, a combination of antibodies reduce the inflammationin an animal by at least 99%, at least 95%, at least 90%, at least 85%,at least 80%, at least 75%, at least 70%, at least 60%, at least 50%, atleast 45%, at least 40%, at least 45%, at least 35%, at least 30%, atleast 25%, at least 20%, or at least 10% relative to the inflammation inan animal in not administered said antibodies. Examples of inflammatorydisorders include, but are not limited to, rheumatoid arthritis,spondyloarthropathies, inflammatory bowel disease and asthma.

[0138] In certain embodiments, the antibody used for treatment ofinflammation (or cancer) is a modified anti-α_(v)β₃ antibody, preferablya Vitaxin antibody (see, PCT publications WO 98/33919 and WO 00/78815,both by Huse et al., and both of which are incorporated by referenceherein in their entireties).

[0139] Antibodies can also be used to prevent the rejection oftransplants. Antibodies can also be used to prevent clot formation.Further, antibodies that function as agonists of the immune response canalso be administered to an animal, preferably a mammal, and mostpreferably a human, to treat, prevent or ameliorate one or more symptomsassociated with the disease, disorder, or infection.

[0140] One or more antibodies that immunospecifically bind to one ormore antigens may be used locally or systemically in the body as atherapeutic. The antibodies of this invention may also be advantageouslyutilized in combination with other monoclonal or chimeric antibodies, orwith lymphokines or hematopoietic growth factors (such as, e.g., IL-2,IL-3 and IL-7), which, for example, serve to increase the number oractivity of effector cells which interact with the antibodies. Theantibodies of this invention may also be advantageously utilized incombination with other monoclonal or chimeric antibodies, or withlymphokines or hematopoietic growth factors (such as, e.g., IL-2, IL-3and IL-7), which, for example, serve to increase the immune response.The antibodies of this invention may also be advantageously utilized incombination with one or more drugs used to treat a disease, disorder, orinfection such as, for example anti-cancer agents, anti-inflammatoryagents or anti-viral agents. Examples of anti-cancer agents include, butare not limited to, isplatin, ifosfamide, paclitaxel, taxanes,topoisomerase I inhibitors (e.g. CPT-11, topotecan, 9-AC, and GG-211),gemcitabine, vinorelbine, oxaliplatin, 5-fluorouracil (5-FU),leucovorin, vinorelbine, temodal, and taxol. Examples of anti-viralagents include, but are not limited to, cytokines (e.g., IFN-α, IFN-β,IFN-γ), inhibitors of reverse transcriptase (e.g., AZT, 3TC, D4T, ddC,ddI, d4T, 3TC, adefovir, efavirenz, delavirdine, nevirapine, abacavir,and other dideoxynucleosides or dideoxyfluoronucleosides), inhibitors ofviral mRNA capping, such as ribavirin, inhibitors of proteases such HIVprotease inhibitors (e.g., amprenavir, indinavir, nelfinavir, ritonavir,and saquinavir,), amphotericin B, castanospermine as an inhibitor ofglycoprotein processing, inhibitors of neuraminidase such as influenzavirus neuraminidase inhibitors (e.g., zanamivir and oseltamivir),topoisomerase I inhibitors (e.g., camptothecins and analogs thereof),amantadine, and rimantadine. Examples of anti-inflammatory agentsinclude, but are not limited to, nonsteroidal anti-inflammatory drugssuch as COX-2 inhibitors (e.g., meloxicam, celecoxib, rofecoxib,flosulide, and SC-58635, and MK-966), ibuprofen and indomethacin, andsteroids (e.g., deflazacort, dexamethasone and methylprednisolone).

[0141] In a specific embodiment, antibodies administered to an animalare of a species origin or species reactivity that is the same speciesas that of the animal. Thus, in a preferred embodiment, human orhumanized antibodies, or nucleic acids encoding human or human, areadministered to a human patient for therapy or prophylaxis.

[0142] In preferred embodiments, immunoglobulins having extended in vivohalf-lives are used in passive immunotherapy (for either therapy orprophylaxis). Because of the extended half-life, passive immunotherapyor prophylaxis can be accomplished using lower doses and/or lessfrequent administration of the therapeutic resulting in fewer sideeffects, better patient compliance, less costly therapy/prophylaxis,etc. In a preferred embodiment, the therapeutic/prophylactic is anantibody that binds RSV, for example, SYNAGIS® or other anti-RSVantibody. Such anti-RSV antibodies, and methods of administration aredisclosed in U.S. patent application Ser. Nos. 09/724,396 and09/724,531, both entitled “Methods of Administering/Dosing Anti-RSVAntibodies For Prophylaxis and Treatment,” both by Young et al., bothfiled Nov. 28, 2000, and continuation-in-part applications of theseapplications, Ser. Nos. ______ and ______, respectively (attorney docketNos. 10271-048 and 10271-047, respectively), also entitled “Methods ofAdministering/Dosing Anti-RSV Antibodies for Prophylaxis and Treatment,”by Young et al., all which are incorporated by reference herein in theirentireties. Also included are the anti-RSV antibodies described inSection 5.1, supra.

[0143] In a specific embodiment, fusion proteins administered to ananimal are of a species origin or species reactivity that is the samespecies as that of the animal. Thus, in a preferred embodiment, humanfusion proteins or nucleic acids encoding human fusion proteins, areadministered to a human subject for therapy or prophylaxis.

5.6. PROPHYLACTIC AND THERAPEUTIC USES OF FUSION PROTEINS AND CONJUGATEDMOLECULES

[0144] The present invention encompasses fusion protein-based andconjugated molecule-based therapies which involve administering fusionproteins or conjugated molecules to an animal, preferably a mammal andmost preferably a human, for preventing, treating, or amelioratingsymptoms associated with a disease, disorder, or infection. Prophylacticand therapeutic compounds of the invention include, but are not limitedto, fusion proteins and nucleic acids encoding fusion proteins andconjugated molecules. Fusion proteins and conjugated molecules may beprovided in pharmaceutically acceptable compositions as known in the artor as described herein.

[0145] Fusion proteins and conjugated molecules of the present inventionthat function as antagonists of a disease, disorder, or infection can beadministered to an animal, preferably a mammal, and most preferably ahuman, to treat, prevent or ameliorate one or more symptoms associatedwith the disease, disorder, or infection. Further, fusion proteins andconjugated molecules of the present invention that function as agonistsof the immune response may be administered to an animal, preferably amammal, and most preferably a human, to treat, prevent or ameliorate oneor more symptoms associated with the disease, disorder, or infection.

[0146] One or more fusion proteins and conjugated molecules may be usedlocally or systemically in the body as a therapeutic. The fusionproteins and conjugated molecules of this invention may also beadvantageously utilized in combination with monoclonal or chimericantibodies, or with lymphokines or hematopoietic growth factors (suchas, e.g., IL-2, IL-3 and IL-7), which, for example, serve to increasethe number or activity of effector cells which interact with theantibodies. The fusion proteins and conjugated molecules of thisinvention may also be advantageously utilized in combination withmonoclonal or chimeric antibodies, or with lymphokines or hematopoieticgrowth factors (such as, e.g., IL-2, IL-3 and IL-7), which, for example,serve to increase the immune response. The fusion proteins andconjugated molecules of this invention may also be advantageouslyutilized in combination with one or more drugs used to treat a disease,disorder, or infection such as, for example anti-cancer agents,anti-inflammatory agents or anti-viral agents. Examples of anti-canceragents include, but are not limited to, isplatin, ifosfamide,paclitaxel, taxanes, topoisomerase I inhibitors (e.g., CPT-11,topotecan, 9-AC, and GG-211), gemcitabine, vinorelbine, oxaliplatin,5-fluorouracil (5-FU), leucovorin, vinorelbine, temodal, and taxol.Examples of anti-viral agents include, but are not limited to, cytokines(e.g, IFN-α, IFN-β, IFN-γ), inhibitors of reverse transcriptase (e.g.,AZT, 3TC, D4T, ddC, ddI, d4T, 3TC, adefovir, efavirenz, delavirdine,nevirapine, abacavir, and other dideoxynucleosides ordideoxyfluoronucleosides), inhibitors of viral mRNA capping, such asribavirin, inhibitors of proteases such HIV protease inhibitors (e.g.,amprenavir, indinavir, nelfinavir, ritonavir, and saquinavir,),amphotericin B, castanospermine as an inhibitor of glycoproteinprocessing, inhibitors of neuraminidase such as influenza virusneuraminidase inhibitors (e.g., zanamivir and oseltamivir),topoisomerase I inhibitors (e.g., camptothecins and analogs thereof),amantadine, and rimantadine. Examples of anti-inflammatory agentsinclude, but are not limited to, nonsteroidal anti-inflammatory drugssuch as COX-2 inhibitors (e.g., meloxicam, celecoxib, rofecoxib,flosulide, and SC-58635, and MK-966), ibuprofen and indomethacin, andsteroids (e.g., deflazacort, dexamethasone and methylprednisolone).

5.7. ADMINISTRATION OF ANTIBODIES OR FUSION PROTEINS

[0147] The invention provides methods of treatment, prophylaxis, andamelioration of one or more symptoms associated with a disease, disorderor infection by administrating to a subject of an effective amount of anantibody of the invention, or pharmaceutical composition comprising anantibody of the invention. The invention also provides methods oftreatment, prophylaxis, and amelioration of one or more symptomsassociated with a disease, disorder or infection by administering to asubject an effective amount of a fusion protein or conjugated moleculeof the invention, or a pharmaceutical composition comprising a fusionprotein or conjugated molecules of the invention. In a preferred aspect,an antibody or fusion protein or conjugated molecule, is substantiallypurified (i.e., substantially free from substances that limit its effector produce undesired side-effects). In a specific embodiment, thesubject is an animal, preferably a mammal such as non-primate (e.g.,cows, pigs, horses, cats, dogs, rats etc.) and a primate (e.g., monkeysuch as a cynomolgous monkey and a human). In a preferred embodiment,the subject is a human.

[0148] Various delivery systems are known and can be used to administeran antibody or fusion protein or conjugated molecule of the invention,e.g., encapsulation in liposomes, microparticles, microcapsules,recombinant cells capable of expressing the antibody or fusion protein,receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem.,262:4429-4432, 1987), construction of a nucleic acid as part of aretroviral or other vector, etc. Methods of administering an antibody, afusion protein or conjugated molecule, or pharmaceutical compositioninclude, but are not limited to, parenteral administration (e.g.,intradermal, intramuscular, intraperitoneal, intravenous andsubcutaneous), epidural, and mucosal (e.g., intranasal and oral routes).In a specific embodiment, antibodies, fusion proteins, conjugatedmolecules, or pharmaceutical compositions are administeredintramuscularly, intravenously, or subcutaneously. The compositions maybe administered by any convenient route, for example by infusion orbolus injection, by absorption through epithelial or mucocutaneouslinings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and maybe administered together with other biologically active agents.Administration can be systemic or local. In addition, pulmonaryadministration can also be employed, e.g., by use of an inhaler ornebulizer, and formulation with an aerosolizing agent. See, e.g., U.S.Pat. Nos. 6,019,968; 5,985, 320; 5,985,309; 5,934,272; 5,874,064;5,855,913; 5,290,540; and 4,880,078; and PCT Publication Nos. WO92/19244; WO 97/32572; WO 97/44013; WO 98/31346; and WO 99/66903, eachof which is incorporated herein by reference in its entirety. In apreferred embodiment, an antibody, a fusion protein, conjugatedmolecules, or a pharmaceutical composition is administered usingAlkermes AIR™ pulmonary drug delivery technology (Alkermes, Inc.,Cambridge, Mass.).

[0149] The invention also provides that an antibody, a fusion protein,or conjugated molecule is packaged in a hermetically sealed containersuch as an ampoule or sachette indicating the quantity of antibody,fusion protein, or conjugated molecule. In one embodiment, the antibody,fusion protein, or conjugated molecule is supplied as a dry sterilizedlyophilized powder or water free concentrate in a hermetically sealedcontainer and can be reconstituted, e.g., with water or saline to theappropriate concentration for administration to a subject. Preferably,the antibody, fusion protein, or conjugated molecule is supplied as adry sterile lyophilized powder in a hermetically sealed container at aunit dosage of at least 5 mg, more preferably at least 10 mg, at least15 mg, at least 25 mg, at least 35 mg, at least 45 mg, at least 50 mg,or at least 75 mg. The lyophilized antibody, fusion protein, orconjugated molecule should be stored at between 2 and 8° C. in itsoriginal container and the antibody, fusion protein, or conjugatedmolecules should be administered within 12 hours, preferably within 6hours, within 5 hours, within 3 hours, or within 1 hour after beingreconstituted. In an alternative embodiment, an antibody, fusionprotein, or conjugated molecule is supplied in liquid form in ahermetically sealed container indicating the quantity and concentrationof the antibody, fusion protein, or conjugated molecule. Preferably, theliquid form of the antibody, fusion protein, or conjugated molecule issupplied in a hermetically sealed container at least 1 mg/ml, morepreferably at least 2.5 mg/ml, at least 5 mg/ml, at least 8 mg/ml, atleast 10 mg/ml, at least 15 mg/kg, or at least 25 mg/ml.

[0150] In a specific embodiment, it may be desirable to administer thepharmaceutical compositions of the invention locally to the area in needof treatment; this may be achieved by, for example, and not by way oflimitation, local infusion, by injection, or by means of an implant,said implant being of a porous, non-porous, or gelatinous material,including membranes, such as sialastic membranes, or fibers. Preferably,when administering an antibody or a fusion protein, care must be takento use materials to which the antibody or the fusion protein does notabsorb.

[0151] In another embodiment, the composition can be delivered in avesicle, in particular a liposome (see Langer, Science, 249:1527-1533,1990; Treat et al., in Liposomes in the Therapy of Infectious Diseaseand Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp.353-365 (1989); Lopez-Berestein, ibid., pp. 3 17-327; see generallyibid.).

[0152] In yet another embodiment, the composition can be delivered in acontrolled release or sustained release system. Any technique known toone of skill in the art can be used to produce sustained releaseformulations comprising one or more antibodies, or one or more fusionproteins. See, e.g. U.S. Pat. No. 4,526,938; PCT publication WO91/05548; PCT publication WO 96/20698; Ning et al., “IntratumoralRadioimmunotheraphy of a Human Colon Cancer Xenograft Using aSustained-Release Gel,” Radiotherapy & Oncology, 39:179-189, 1996; Songet al., “Antibody Mediated Lung Targeting of Long-CirculatingEmulsions,” PDA Journal of Pharmaceutical Science & Technology,50:372-397, 1995; Cleek et al., “Biodegradable Polymeric Carriers for abFGF Antibody for Cardiovascular Application,” Pro. Intl. Symp. Control.Rel. Bioact. Mater., 24:853-854, 1997; and Lam et al.,“Microencapsulation of Recombinant Humanized Monoclonal Antibody forLocal Delivery,” Proc. Int'l. Symp. Control Rel. Bioact. Mater.,24:759-760, 1997, each of which is incorporated herein by reference inits entirety. In one embodiment, a pump may be used in a controlledrelease system (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng.,14:20, 1987; Buchwald et al., Surgery, 88:507, 1980; and Saudek et al.,N. Engl. J. Med., 321:574, 1989). In another embodiment, polymericmaterials can be used to achieve controlled release of antibodies orfusion proteins (see e.g., Medical Applications of Controlled Release,Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); ControlledDrug Bioavailability, Drug Product Design and Performance, Smolen andBall (eds.), Wiley, N.Y. (1984); Ranger and Peppas, J., Macromol. Sci.Rev. Macromol. Chem., 23:61, 1983; see also Levy et al., Science,228:190, 1985; During et al., Ann. Neurol., 25:351, 1989; Howard et al.,J. Neurosurg., 7 1:105, 1989); U.S. Pat. No. 5,679,377; U.S. Pat. No.5,916,597; U.S. Pat. No. 5,912,015; U.S. Pat. No. 5,989,463; U.S. Pat.No. 5,128,326; PCT Publication No. WO 99/15154; and PCT Publication No.WO 99/20253). In yet another embodiment, a controlled release system canbe placed in proximity of the therapeutic target (e.g., the lungs), thusrequiring only a fraction of the systemic dose (see, e.g., Goodson, inMedical Applications of Controlled Release, supra, vol. 2, pp. 115-138(1984)).

[0153] Other controlled release systems are discussed in the review byLanger, Science, 249:1527-1533, 1990).

[0154] In a specific embodiment where the composition of the inventionis a nucleic acid encoding an antibody or fusion protein, the nucleicacid can be administered in vivo to promote expression of its encodedantibody or fusion protein, by constructing it as part of an appropriatenucleic acid expression vector and administering it so that it becomesintracellular, e.g., by use of a retroviral vector (see U.S. Pat. No.4,980,286), or by direct injection, or by use of microparticlebombardment (e.g., a gene gun; Biolistic, Dupont), or coating withlipids or cell-surface receptors or transfecting agents, or byadministering it in linkage to a homeobox-like peptide which is known toenter the nucleus (see e.g., Joliot et al., Proc. Natl. Acad. Sci. USA,88:1864-1868, 1991), etc. Alternatively, a nucleic acid can beintroduced intracellularly and incorporated within host cell DNA forexpression by homologous recombination.

[0155] The present invention also provides pharmaceutical compositions.Such compositions comprise a prophylactically or therapeuticallyeffective amount of an antibody, fusion protein or conjugated molecule,and a pharmaceutically acceptable carrier. In a specific embodiment, theterm “pharmaceutically acceptable” means approved by a regulatory agencyof the Federal or a state government or listed in the U.S. Pharmacopeiaor other generally recognized pharmacopeia for use in animals, and moreparticularly in humans. The term “carrier” refers to a diluent, adjuvant(e.g., Freund's complete and incomplete, mineral gels such as aluminumhydroxide, surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanins, dinitrophenol, and potentially useful adjuvants for humanssuch as BCG (Bacille Calmette-Guerin) and Corynebacterium parvum),excipient, or vehicle with which the therapeutic is administered. Suchpharmaceutical carriers can be sterile liquids, such as water and oils,including those of petroleum, animal, vegetable or synthetic origin,such as peanut oil, soybean oil, mineral oil, sesame oil and the like.Water is a preferred carrier when the pharmaceutical composition isadministered intravenously. Saline solutions and aqueous dextrose andglycerol solutions can also be employed as liquid carriers, particularlyfor injectable solutions. Suitable pharmaceutical excipients includestarch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk,silica gel, sodium stearate, glycerol monostearate, talc, sodiumchloride, dried skim milk, glycerol, propylene, glycol, water, ethanoland the like. The composition, if desired, can also contain minoramounts of wetting or emulsifying agents, or pH buffering agents. Thesecompositions can take the form of solutions, suspensions, emulsion,tablets, pills, capsules, powders, sustained-release formulations andthe like. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin. Such compositions will containa prophylactically or therapeutically effective amount of the antibodyor fragment thereof, or fusion protein or conjugated molecule,preferably in purified form, together with a suitable amount of carrierso as to provide the form for proper administration to the patient. Theformulation should suit the mode of administration.

[0156] In a preferred embodiment, the composition is formulated inaccordance with routine procedures as a pharmaceutical compositionadapted for intravenous administration to human beings. Typically,compositions for intravenous administration are solutions in sterileisotonic aqueous buffer. Where necessary, the composition may alsoinclude a solubilizing agent and a local anesthetic such as lignocaineto ease pain at the site of the injection.

[0157] Generally, the ingredients of compositions of the invention aresupplied either separately or mixed together in unit dosage form, forexample, as a dry lyophilized powder or water free concentrate in ahermetically sealed container such as an ampoule or sachette indicatingthe quantity of active agent. Where the composition is to beadministered by infusion, it can be dispensed with an infusion bottlecontaining sterile pharmaceutical grade water or saline. Where thecomposition is administered by injection, an ampoule of sterile waterfor injection or saline can be provided so that the ingredients may bemixed prior to administration.

[0158] The compositions of the invention can be formulated as neutral orsalt forms. Pharmaceutically acceptable salts include those formed withanions such as those derived from hydrochloric, phosphoric, acetic,oxalic, tartaric acids, etc., and those formed with cations such asthose derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

[0159] The amount of the composition of the invention which will beeffective in the treatment, prevention or amelioration of one or moresymptoms associated with a disease, disorder, or infection can bedetermined by standard clinical techniques. The precise dose to beemployed in the formulation will depend on the route of administration,the age of the subject, and the seriousness of the disease, disorder, orinfection, and should be decided according to the judgment of thepractitioner and each patient's circumstances. Effective doses may beextrapolated from dose-response curves derived from in vitro or animalmodel (e.g., the cotton rat or Cynomolgous monkey) test systems.

[0160] For fusion proteins, the therapeutically or prophylacticallyeffective dosage administered to a subject ranges from about 0.001 to 50mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, morepreferably about 0.1 to 20 mg/kg body weight, and even more preferablyabout 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6mg/kg body weight. For antibodies, the therapeutically orprophylactically effective dosage administered to a subject is typically0.1 mg/kg to 200 mg/kg of the subject's body weight. Preferably, thedosage administered to a subject is between 0.1 mg/kg and 20 mg/kg ofthe subject's body weight and more preferably the dosage administered toa subject is between 1 mg/kg to 10 mg/kg of the subject's body weight.The dosage will, however, depend upon the extent to which the in vivohalf-life of the molecule has been increased Generally, human antibodiesand human fusion proteins have longer half-lives within the human bodythan antibodies of fusion proteins from other species due to the immuneresponse to the foreign polypeptides. Thus, lower dosages of humanantibodies or human fusion proteins and less frequent administration isoften possible. Further, the dosage and frequency of administration ofantibodies, fusion proteins, or conjugated molecules may be reduced alsoby enhancing uptake and tissue penetration (e.g., into the lung) of theantibodies or fusion proteins by modifications such as, for example,lipidation.

[0161] Treatment of a subject with a therapeutically or prophylacticallyeffective amount of an antibody, fusion protein, or conjugated moleculecan include a single treatment or, preferably, can include a series oftreatments. In a preferred example, a subject is treated with anantibody, fusion protein, or conjugated molecule in the range of betweenabout 0.1 to 30 mg/kg body weight, one time per week for between about 1to 10 weeks, preferably between 2 to 8 weeks, more preferably betweenabout 3 to 7 weeks, and even more referably for about 4, 5, or 6 weeks.In other embodiments, the pharmaceutical composition of the invention isadministered once a day, twice a day, or three times a day. In otherembodiments, the pharmaceutical composition is administered once a week,twice a week, once every two weeks, once a month, once every six weeks,once every two months, twice a year or once per year. It will also beappreciated that the effective dosage of the antibody, fusion protein,or conjugated molecule used for treatment may increase or decrease overthe course of a particular treatment.

5.7.1. GENE THERAPY

[0162] In a specific embodiment, nucleic acids comprising sequencesencoding antibodies or fusion proteins, are administered to treat,prevent or ameliorate one or more symptoms associated with a disease,disorder, or infection, by way of gene therapy. Gene therapy refers totherapy performed by the administration to a subject of an expressed orexpressible nucleic acid. In this embodiment of the invention, thenucleic acids produce their encoded antibody or fusion protein thatmediates a therapeutic or prophylactic effect.

[0163] Any of the methods for gene therapy available in the art can beused according to the present invention. Exemplary methods are describedbelow.

[0164] For general reviews of the methods of gene therapy, see Goldspielet al., Clinical Pharmacy, 12:488-505, 1993; Wu and Wu, Biotherapy,3:87-95, 1991; Tolstoshev, Ann. Rev. Pharmacol. Toxicol., 32:573-596,1993; Mulligan, Science, 260:926-932, 1993; and Morgan and Anderson,Ann. Rev. biochem. 62:191-217, 1993; TIBTECH 11(5):155-215, 1993.Methods commonly known in the art of recombinant DNA technology whichcan be used are described in Ausubel et al. (eds.), Current Protocols inMolecular Biology, John Wiley & Sons, NY (1993); and Kriegler, GeneTransfer and Expression, A Laboratory Manual, Stockton Press, NY (1990).

[0165] In a preferred aspect, a composition of the invention comprisesnucleic acids encoding an antibody, said nucleic acids being part of anexpression vector that expresses the antibody in a suitable host. Inparticular, such nucleic acids have promoters, preferably heterologouspromoters, operably linked to the antibody coding region, said promoterbeing inducible or constitutive, and, optionally, tissue-specific. Inanother particular embodiment, nucleic acid molecules are used in whichthe antibody coding sequences and any other desired sequences areflanked by regions that promote homologous recombination at a desiredsite in the genome, thus providing for intrachromosomal expression ofthe antibody encoding nucleic acids (Koller and Smithies, Proc. Natl.Acad. Sci. USA, 86:8932-8935, 1989; and Zijlstra et al., Nature,342:435-438, 1989).

[0166] In another preferred aspect, a composition of the inventioncomprises nucleic acids encoding a fusion protein, said nucleic acidsbeing a part of an expression vector that expression the fusion proteinin a suitable host. In particular, such nucleic acids have promoters,preferably heterologous promoters, operably linked to the coding regionof a fusion protein, said promoter being inducible or constitutive, andoptionally, tissue-specific. In another particular embodiment, nucleicacid molecules are used in which the coding sequence of the fusionprotein and any other desired sequences are flanked by regions thatpromote homologous recombination at a desired site in the genome, thusproviding for intrachromosomal expression of the fusion protein encodingnucleic acids.

[0167] Delivery of the nucleic acids into a subject may be eitherdirect, in which case the subject is directly exposed to the nucleicacid or nucleic acid-carrying vectors, or indirect, in which case, cellsare first transformed with the nucleic acids in vitro, then transplantedinto the subject. These two approaches are known, respectively, as invivo or ex vivo gene therapy.

[0168] In a specific embodiment, the nucleic acid sequences are directlyadministered in vivo, where it is expressed to produce the encodedproduct. This can be accomplished by any of numerous methods known inthe art, e.g., by constructing them as part of an appropriate nucleicacid expression vector and administering it so that they becomeintracellular, e.g., by infection using defective or attenuatedretroviral or other viral vectors (see U.S. Pat. No. 4,980,286), or bydirect injection of naked DNA, or by use of microparticle bombardment(e.g., a gene gun; Biolistic, Dupont), or coating with lipids orcell-surface receptors or transfecting agents, encapsulation inliposomes, microparticles, or microcapsules, or by administering them inlinkage to a peptide which is known to enter the nucleus, byadministering it in linkage to a ligand subject to receptor-mediatedendocytosis (see, e.g., Wu and Wu, J. Biol. Chem., 262:4429-4432, 1987)(which can be used to target cell types specifically expressing thereceptors), etc. In another embodiment, nucleic acid-ligand complexescan be formed in which the ligand comprises a fusogenic viral peptide todisrupt endosomes, allowing the nucleic acid to avoid lysosomaldegradation. In yet another embodiment, the nucleic acid can be targetedin vivo for cell specific uptake and expression, by targeting a specificreceptor (see, e.g., PCT Publications WO 92/06180; WO 92/22635; WO92/20316; WO 93/14188; WO 93/20221). Alternatively, the nucleic acid canbe introduced intracellularly and incorporated within host cell DNA forexpression, by homologous recombination (Koller and Smithies, Proc.Natl. Acad. Sci. USA, 86:8932-8935, 1989; and Zijlstra et al., Nature,342:435-438, 1989).

[0169] In a specific embodiment, viral vectors that contain nucleic acidsequences encoding an antibody or a fusion protein are used. Forexample, a retroviral vector can be used (see Miller et al., Meth.Enzymol., 217:581-599, 1993). These retroviral vectors contain thecomponents necessary for the correct packaging of the viral genome andintegration into the host cell DNA. The nucleic acid sequences encodingthe antibody or a fusion protein to be used in gene therapy are clonedinto one or more vectors, which facilitates delivery of the nucleotidesequence into a subject. More detail about retroviral vectors can befound in Boesen et al., Biotherapy, 6:291-302, 1994, which describes theuse of a retroviral vector to deliver the mdr 1 gene to hematopoieticstem cells in order to make the stem cells more resistant tochemotherapy. Other references illustrating the use of retroviralvectors in gene therapy are: Clowes et al., J. Clin. Invest.,93:644-651, 1994; Klein et al., Blood 83:1467-1473, 1994; Salmons andGunzberg, Human Gene Therapy, 4:129-141, 1993; and Grossman and Wilson,Curr. Opin. in Genetics and Devel., 3:110-114, 1993.

[0170] Adenoviruses are other viral vectors that can be used in genetherapy. Adenoviruses are especially attractive vehicles for deliveringgenes to respiratory epithelia. Adenoviruses naturally infectrespiratory epithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Kozarsky and Wilson, CurrentOpinion in Genetics and Development, 3:499-503, 1993, present a reviewof adenovirus-based gene therapy. Bout et al., Human Gene Therapy,5:3-10, 1994, demonstrated the use of adenovirus vectors to transfergenes to the respiratory epithelia of rhesus monkeys. Other instances ofthe use of adenoviruses in gene therapy can be found in Rosenfeld etal., Science, 252:431-434, 1991; Rosenfeld et al., Cell, 68:143-155,1992; Mastrangeli et al., J. Clin. Invest., 91:225-234, 1993; PCTPublication WO 94/12649; and Wang et al., Gene Therapy, 2:775-783, 1995.In a preferred embodiment, adenovirus vectors are used.

[0171] Adeno-associated virus (AAV) has also been proposed for use ingene therapy (see, e.g.,Walsh et al., Proc. Soc. Exp. Biol. Med.,204:289-300, 1993, and U.S. Pat. No. 5,436,146).

[0172] Another approach to gene therapy involves transferring a gene tocells in tissue culture by such methods as electroporation, lipofection,calcium phosphate mediated transfection, or viral infection. Usually,the method of transfer includes the transfer of a selectable marker tothe cells. The cells are then placed under selection to isolate thosecells that have taken up and are expressing the transferred gene. Thosecells are then delivered to a subject.

[0173] In this embodiment, the nucleic acid is introduced into a cellprior to administration in vivo of the resulting recombinant cell. Suchintroduction can be carried out by any method known in the art,including but not limited to transfection, electroporation,microinjection, infection with a viral or bacteriophage vectorcontaining the nucleic acid sequences, cell fusion, chromosome-mediatedgene transfer, microcellmediated gene transfer, spheroplast fusion, etc.Numerous techniques are known in the art for the introduction of foreigngenes into cells (see, e.g., Loeffler and Behr, Meth. Enzymol.,217:599-618, 1993; Cohen et al., Meth. Enzymol., 217:618-644, 1993; andClin. Pharma. Ther., 29:69-92, 1985) and may be used in accordance withthe present invention, provided that the necessary developmental andphysiological functions of the recipient cells are not disrupted. Thetechnique should provide for the stable transfer of the nucleic acid tothe cell, so that the nucleic acid is expressible by the cell andpreferably heritable and expressible by its cell progeny.

[0174] The resulting recombinant cells can be delivered to a subject byvarious methods known in the art. Recombinant blood cells (e.g.,hematopoietic stem or progenitor cells) are preferably administeredintravenously. The amount of cells envisioned for use depends on thedesired effect, patient state, etc., and can be determined by oneskilled in the art.

[0175] Cells into which a nucleic acid can be introduced for purposes ofgene therapy encompass any desired, available cell type, and include butare not limited to epithelial cells, endothelial cells, keratinocytes,fibroblasts, muscle cells, hepatocytes; blood cells such as Tlymphocytes, B lymphocytes, monocytes, macrophages, neutrophils,eosinophils, megakaryocytes, granulocytes; various stem or progenitorcells, in particular hematopoietic stem or progenitor cells, e.g., asobtained from bone marrow, umbilical cord blood, peripheral blood, fetalliver, etc.

[0176] In a preferred embodiment, the cell used for gene therapy isautologous to the subject.

[0177] In an embodiment in which recombinant cells are used in genetherapy, nucleic acid sequences encoding an antibody or a fusion proteinare introduced into the cells such that they are expressible by thecells or their progeny, and the recombinant cells are then administeredin vivo for therapeutic effect. In a specific embodiment, stem orprogenitor cells are used. Any stem and/or progenitor cells which can beisolated and maintained in vitro can potentially be used in accordancewith this embodiment of the present invention (see e.g., PCT PublicationWO 94/08598; Stemple and Anderson, Cell, 7 1:973-985, 1992; Rheinwald,Meth. Cell Bio., 21A:229, 1980; and Pittelkow and Scott, Mayo ClinicProc., 61:771, 1986).

[0178] In a specific embodiment, the nucleic acid to be introduced forpurposes of gene therapy comprises an inducible promoter operably linkedto the coding region, such that expression of the nucleic acid iscontrollable by controlling the presence or absence of the appropriateinducer of transcription.

5.8. CHARACTERIZATION AND DEMONSTRATION OF THERAPEUTIC OR PROPHYLACTICUTILITY

[0179] Antibodies, fusion proteins, and conjugated molecules of thepresent invention may be characterized in a variety of ways. Inparticular, antibodies of the invention may be assayed for the abilityto immunospecifically bind to an antigen. Such an assay may be performedin solution (e.g., Houghten, Bio/Techniques, 13:412-421, 1992), on beads(Lam, Nature, 354:82-84, 1991, on chips (Fodor, Nature, 364:555-556,1993), on bacteria (U.S. Pat. No. 5,223,409), on spores (U.S. Pat. Nos.5,571,698; 5,403,484; and 5,223,409), on plasmids (Cull et al., Proc.Natl. Acad. Sci. USA, 89:1865-1869, 1992) or on phage (Scott and Smith,Science, 249:386-390, 1990; Devlin, Science, 249:404-406, 1990; Cwirlaet al., Proc. Natl. Acad. Sci. USA, 87:6378-6382, 1990; and Felici, J.Mol. Biol., 222:301-310, 1991) (each of these references is incorporatedherein in its entirety by reference). Antibodies that have beenidentified to immunospecifically bind to an antigen or a fragmentthereof can then be assayed for their specificity affinity for theantigen.

[0180] The antibodies of the invention or fragments thereof may beassayed for immunospecific binding to an antigen and cross-reactivitywith other antigens by any method known in the art. Immunoassays whichcan be used to analyze immunospecific binding and cross-reactivityinclude, but are not limited to, competitive and non-competitive assaysystems using techniques such as western blots, radioimmunoassays, ELISA(enzyme linked immunosorbent assay), “sandwich” immunoassays,immunoprecipitation assays, precipitin reactions, gel diffusionprecipitin reactions, immunodiffusion assays, agglutination assays,complement-fixation assays, immunoradiometric assays, fluorescentimmunoassays, protein A immunoassays, to name but a few. Such assays areroutine and well known in the art (see, e.g, Ausubel et al, eds, 1994,Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc.,New York, which is incorporated by reference herein in its entirety).Exemplary immunoassays are described briefly below (but are not intendedby way of limitation).

[0181] Immunoprecipitation protocols generally comprise lysing apopulation of cells in a lysis buffer such as RIPA buffer (1% NP-40 orTriton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 Msodium phosphate at pH 7.2, 1% Trasylol) supplemented with proteinphosphatase and/or protease inhibitors (e.g., EDTA, PMSF, aprotinin,sodium vanadate), adding the antibody of interest to the cell lysate,incubating for a period of time (e.g., 1 to 4 hours) at 40° C., addingprotein A and/or protein G sepharose beads to the cell lysate,incubating for about an hour or more at 40° C., washing the beads inlysis buffer and resuspending the beads in SDS/sample buffer. Theability of the antibody of interest to immunoprecipitate a particularantigen can be assessed by, e.g., western blot analysis. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the binding of the antibody to an antigen and decrease thebackground (e.g., pre-clearing the cell lysate with sepharose beads).For further discussion regarding immunoprecipitation protocols see,e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology,Vol. 1, John Wiley & Sons, Inc., New York at 10.16.1.

[0182] Western blot analysis generally comprises preparing proteinsamples, electrophoresis of the protein samples in a polyacrylamide gel(e.g., 8%-20% SDS-PAGE depending on the molecular weight of theantigen), transferring the protein sample from the polyacrylamide gel toa membrane such as nitrocellulose, PVDF or nylon, blocking the membranein blocking solution (e.g., PBS with 3% BSA or non-fat milk), washingthe membrane in washing buffer (e.g., PBS-Tween 20), blocking themembrane with primary antibody (the antibody of interest) diluted inblocking buffer, washing the membrane in washing buffer, blocking themembrane with a secondary antibody (which recognizes the primaryantibody, e.g., an anti-human antibody) conjugated to an enzymaticsubstrate (e.g., horseradish peroxidase or alkaline phosphatase) orradioactive molecule (e.g., ³²P or ¹²⁵I) diluted in blocking buffer,washing the membrane in wash buffer, and detecting the presence of theantigen. One of skill in the art would be knowledgeable as to theparameters that can be modified to increase the signal detected and toreduce the background noise. For further discussion regarding westernblot protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols inMolecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.8.1.

[0183] ELISAs comprise preparing antigen, coating the well of a 96 wellmicrotiter plate with the antigen, adding the antibody of interestconjugated to a detectable compound such as an enzymatic substrate(e.g., horseradish peroxidase or alkaline phosphatase) to the well andincubating for a period of time, and detecting the presence of theantigen. In ELISAs the antibody of interest does not have to beconjugated to a detectable compound; instead, a second antibody (whichrecognizes the antibody of interest) conjugated to a detectable compoundmay be added to the well. Further, instead of coating the well with theantigen, the antibody may be coated to the well. In this case, a secondantibody conjugated to a detectable compound may be added following theaddition of the antigen of interest to the coated well. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected as well as other variations of ELISAsknown in the art. For further discussion regarding ELISAs see, e.g.,Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol.1, John Wiley & Sons, Inc., New York at 11.2.1.

[0184] The binding affinity of an antibody to an antigen and theoff-rate of an antibody-antigen interaction can be determined bycompetitive binding assays. One example of a competitive binding assayis a radioimmunoassay comprising the incubation of labeled antigen(e.g., ³H or ¹²⁵I) with the antibody of interest in the presence ofincreasing amounts of unlabeled antigen, and the detection of theantibody bound to the labeled antigen. The affinity of the antibody ofthe present invention or a fragment thereof for the antigen and thebinding off-rates can be determined from the saturation data byscatchard analysis. Competition with a second antibody can also bedetermined using radioimmunoassays. In this case, the antigen isincubated with an antibody of the present invention or a fragmentthereof conjugated to a labeled compound (e.g., ³H or ¹²⁵I) in thepresence of increasing amounts of an unlabeled second antibody.

[0185] In a preferred embodiment, BIAcore kinetic analysis is used todetermine the binding on and off rates of antibodies to an antigen.BIAcore kinetic analysis comprises analyzing the binding anddissociation of an antigen from chips with immobilized antibodies ontheir surface (see the Example section infra).

[0186] The antibodies of the invention as well as fusion proteins andconjugated molecules can also be assayed for their ability to inhibitthe binding of an antigen to its host cell receptor using techniquesknown to those of skill in the art. For example, cells expressing thereceptor for a viral antigen can be contacted with virus in the presenceor absence of an antibody and the ability of the antibody to inhibitviral antigen's binding can measured by, for example, flow cytometry ora scintillation counter. The antigen or the antibody can be labeled witha detectable compound such as a radioactive label (e.g. ³²P, ³⁵S, and¹²⁵I) or a fluorescent label (e.g., fluorescein isothiocyanate,rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehydeand fluorescamine) to enable detection of an interaction between theantigen and its host cell receptor. Alternatively, the ability ofantibodies to inhibit an antigen from binding to its receptor can bedetermined in cell-free assays. For example, virus or a viral antigen(e.g., RSV F glycoprotein) can be contacted in a cell-free assay with anantibody and the ability of the antibody to inhibit the virus or theviral antigen from binding to its host cell receptor can be determined.Preferably, the antibody is immobilized on a solid support and theantigen is labeled with a detectable compound. Alternatively, theantigen is immobilized on a solid support and the antibody is labeledwith a detectable compound. The antigen may be partially or completelypurified (e.g., partially or completely free of other polypeptides) orpart of a cell lysate. Further, the antigen may be a fusion proteincomprising the viral antigen and a domain such asglutathionine-S-transferase. Alternatively, an antigen can bebiotinylated using techniques well known to those of skill in the art(e.g, biotinylation kit, Pierce Chemicals; Rockford, Ill.).

[0187] The antibodies, fusion proteins, and conjugated molecules of theinvention can also be assayed for their ability to inhibit ordownregulate viral or bacterial replication using techniques known tothose of skill in the art. For example, viral replication can be assayedby a plaque assay such as described, e.g., by Johnson et al., Journal ofInfectious Diseases, 176:1215-1224, 1997. The antibodies, fusionproteins, and conjugated molecules of the invention of the invention canalso be assayed for their ability to inhibit or downregulate theexpression of viral or bacterial polypeptides. Techniques known to thoseof skill in the art, including, but not limited to, Western blotanalysis, Northern blot analysis, and RT-PCR, can be used to measure theexpression of viral or bacterial polypeptides. Further, the antibodies,fusion proteins, and conjugated molecules of the invention of theinvention can be assayed for their ability to prevent the formation ofsyncytia.

[0188] The antibodies, fusion proteins, conjugated molecules, andcompositions of the invention are preferably tested in vitro, and thenin vivo for the desired therapeutic or prophylactic activity, prior touse in humans. For example, in vitro assays which can be used todetermine whether administration of a specific antibody, a specificfusion protein, a specific conjugated molecule, or a composition of thepresent invention is indicated, include in vitro cell culture assays inwhich a subject tissue sample is grown in culture, and exposed to orotherwise administered an antibody, a fusion protein, conjugatedmolecule, or composition of the present invention, and the effect ofsuch an antibody, a fusion protein, conjugated molecule, or acomposition of the present invention upon the tissue sample is observed.In various specific embodiments, in vitro assays can be carried out withrepresentative cells of cell types involved in a disease or disorder, todetermine if an antibody, a fusion protein, conjugated molecule, orcomposition of the present invention has a desired effect upon such celltypes. Preferably, the antibodies, the fusion proteins, the conjugatedmolecules, or compositions of the invention are also tested in in vitroassays and animal model systems prior to administration to humans.

[0189] Antibodies, fusion proteins, conjugated molecules, orcompositions of the present invention for use in therapy can be testedfor their toxicity in suitable animal model systems, including but notlimited to rats, mice, cows, monkeys, and rabbits. For in vivo testingfor the toxicity of an antibody, a fusion protein, a conjugatedmolecule, or a composition, any animal model system known in the art maybe used.

[0190] Efficacy in treating or preventing viral infection may bedemonstrated by detecting the ability of an antibody, a fusion protein,a conjugated molecule, or a composition of the invention to inhibit thereplication of the virus, to inhibit transmission or prevent the virusfrom establishing itself in its host, or to prevent, ameliorate oralleviate one or more symptoms associated with viral infection. Thetreatment is considered therapeutic if there is, for example, areduction is viral load, amelioration of one or more symptoms or adecrease in mortality and/or morbidity following administration of anantibody, a fusion protein, a conjugated molecule, or a composition ofthe invention. Antibodies, fusion proteins, conjugated molecules, orcompositions of the invention can also be tested for their ability toinhibit viral replication or reduce viral load in in vitro and in vivoassays.

[0191] Efficacy in treating or preventing bacterial infection may bedemonstrated by detecting the ability of an antibody, a fusion proteinor a composition of the invention to inhibit the bacterial replication,or to prevent, ameliorate or alleviate one or more symptoms associatedwith bacterial infection. The treatment is considered therapeutic ifthere is, for example, a reduction is bacterial numbers, amelioration ofone or more symptoms or a decrease in mortality and/or morbidityfollowing administration of an antibody, a fusion protein or acomposition of the invention.

[0192] Efficacy in treating cancer may be demonstrated by detecting theability of an antibody, a fusion protein, a conjugated molecule, or acomposition of the invention to inhibit or reduce the growth ormetastasis of cancerous cells or to ameliorate or alleviate one or moresymptoms associated with cancer. The treatment is considered therapeuticif there is, for example, a reduction in the growth or metastasis ofcancerous cells, amelioration of one or more symptoms associated withcancer, or a decrease in mortality and/or morbidity followingadministration of an antibody, a fusion protein, a conjugated molecule,or a composition of the invention. Antibodies, fusion proteins orcompositions of the invention can be tested for their ability to reducetumor formation in in vitro, ex vivo, and in vivo assays.

[0193] Efficacy in treating inflammatory disorders may be demonstratedby detecting the ability of an antibody, a fusion protein, a conjugatedmolecule, or a composition of the invention to reduce or inhibit theinflammation in an animal or to ameliorate or alleviate one or moresymptoms associated with an inflammatory disorder. The treatment isconsidered therapeutic if there is, for example, a reduction is ininflammation or amelioration of one or more symptoms followingadministration of an antibody, a fusion proteins, a conjugated molecule,or a composition of the invention.

[0194] Antibodies, fusion proteins, conjugated molecules, orcompositions of the invention can be tested in vitro and in vivo for theability to induce the expression of cytokines (e.g., IFN-α, IFN-β,IFN-γ, IL-2, IL-3, IL-4, IL-5, IL-6, IL10, IL-12, and IL-15) andactivation markers (e.g., CD28, ICOS, and SLAM). Techniques known tothose of skill in the art can be used to measure the level of expressionof cytokines and activation markers. For example, the level ofexpression of cytokines can be measured by analyzing the level of RNA ofcytokines by, for example, RT-PCR and Northern blot analysis, and byanalyzing the level of cytokines by, for example, immunoprecipitationfollowed by Western blot analysis or ELISA.

[0195] Antibodies, fusion proteins, conjugated molecules, orcompositions of the invention can be tested in vitro and in vivo fortheir ability to modulate the biological activity of immune cells,preferably human immune cells (e.g., T-cells, B-cells, and NaturalKiller cells). The ability of an antibody, a fusion protein, aconjugated molecule, or a composition of the invention to modulate thebiological activity of immune cells can be assessed by detecting theexpression of antigens, detecting the proliferation of immune cells,detecting the activation of signaling molecules, detecting the effectorfunction of immune cells, or detecting the differentiation of immunecells. Techniques known to those of skill in the art can be used formeasuring these activities. For example, cellular proliferation can beassayed by ³H-thymidine incorporation assays and trypan blue cellcounts. Antigen expression can be assayed, for example, by immunoassaysincluding, but are not limited to, competitive and non-competitive assaysystems using techniques such as Western blots, immunohistochemistry,radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich”immunoassays, immunoprecipitation assays, precipitin reactions, geldiffusion precipitin reactions, immunodiffusion assays, agglutinationassays, complement-fixation assays, immunoradiometric assays,fluorescent immunoassays, protein A immunoassays and FACS analysis. Theactivation of signaling molecules can be assayed, for example, by kinaseassays and electrophoretic shift assays (EMSAs).

[0196] Antibodies, fusion proteins, conjugated molecules, orcompositions of the invention can also be tested for their ability toincrease the survival period of animals, preferably mammals and mostpreferably humans, suffering from a disease, disorder, or infection byat least 25%, preferably at least 50%, at least 60%, at least 75%, atleast 85%, at least 95%, or at least 99%. Further, antibodies, fusionproteins, conjugated molecules, or compositions of the invention can betested for their ability reduce the hospitalization period of animals,preferably mammals and most preferably humans, suffering from a disease,disorder, or infection by at least 60%, preferably at least 75%, atleast 85%, at least 95%, or at least 99%. Techniques known to those ofskill in the art can be used to analyze the function of the antibodiesor compositions of the invention in vivo.

5.9. DIAGNOSTIC USES OF ANTIBODIES AND FUSION PROTEINS

[0197] Labeled antibodies, fusion proteins, and conjugated molecules ofthe invention can be used for diagnostic purposes to detect, diagnose,or monitor diseases, disorders or infections. The invention provides forthe detection or diagnosis of a disease, disorder or infection,comprising: (a) assaying the expression of an antigen in cells or atissue sample of a subject using one or more antibodies thatimmunospecifically bind to the antigen; and (b) comparing the level ofthe antigen with a control level, e.g, levels in normal tissue samples,whereby an increase in the assayed level of antigen compared to thecontrol level of the antigen is indicative of the disease, disorder orinfection. The invention also provides for the detection or diagnosis ofa disease, disorder or infection, comprising (a) assaying the expressionof an antigen in cells or a tissue sample of a subject using one orfusion proteins or conjugated molecules of the invention that bind tothe antigen; and (b) comparing the level of the antigen with a controllevel, e.g., levels in normal tissue samples, whereby an increase ofantigen compared to the control level of the antigen is indicative ofthe disease, disorder or infection. Accordingly, the fusion protein orconjugated molecule comprises a bioactive molecule such as a ligand,cytokine or growth factor and the hinge-Fc region or fragments thereof,wherein the fusion protein or conjugated molecule is capable of bindingto an antigen being detected.

[0198] Antibodies of the invention can be used to assay antigen levelsin a biological sample using classical immunohistological methods asdescribed herein or as known to those of skill in the art (e.g., seeJalkanen et al., J. Cell. Biol., 101:976-985, 1985; Jalkanen et al., J.Cell. Biol., 105:3087-3096, 1987). Other antibody-based methods usefulfor detecting protein gene expression include immunoassays, such as theenzyme linked immunosorbent assay (ELISA) and the radioimmunoassay(RIA). Suitable antibody assay labels are known in the art and includeenzyme labels, such as, alkaline phosphatase, glucose oxidase;radioisotopes, such as iodine (¹²⁵I, ¹³¹I), carbon (¹⁴C), sulfur (³⁵S),tritium (³H), indium (¹²¹In), and technetium (^(99m)Tc); luminescentlabels, such as luminol; and fluorescent labels, such as fluorescein andrhodamine.

[0199] Fusion proteins can be used to assay antigen levels in abiological sample using, for example, SDS-PAGE and immunoassays known tothose of skill in the art.

[0200] One aspect of the invention is the detection and diagnosis of adisease, disorder, or infection in a human. In one embodiment, diagnosiscomprises: a) administering (for example, parenterally, subcutaneously,or intraperitoneally) to a subject an effective amount of a labeledantibody that immunospecifically binds to an antigen; b) waiting for atime interval following the administration for permitting the labeledantibody to preferentially concentrate at sites in the subject where theantigen is expressed (and for unbound labeled molecule to be cleared tobackground level); c) determining background level; and d) detecting thelabeled antibody in the subject, such that detection of labeled antibodyabove the background level indicates that the subject has the disease,disorder, or infection. In accordance with this embodiment, the antibodyis labeled with an imaging moiety which is detectable using an imagingsystem known to one of skill in the art. Background level can bedetermined by various methods including, comparing the amount of labeledmolecule detected to a standard value previously determined for aparticular system.

[0201] In another embodiment, diagnosis comprises: a) administering (forexample, parenterally, subcutaneously, or intraperitoneally) to asubject an effective amount of a labeled fusion protein or conjugatedmolecule that binds to an antigen or some other molecule; b) waiting fora time interval following the administration for permitting the labeledfusion protein or conjugated molecule to preferentially concentrate atsites in the subject where the antigen or other molecule is expressed(and for unbound labeled molecule to be cleared to background level); c)determining background level; and d) detecting the labeled fusionprotein or conjugated molecule in the subject, such that detection oflabeled fusion protein above the background level indicates that thesubject has the disease, disorder, or infection. In accordance with thisembodiment, the fusion protein or conjugated molecule comprises abioactive molecule such as a ligand, cytokine or growth factor and ahinge-Fc region or a fragment thereof, wherein said fusion protein orconjugated molecule is labeled with an imaging moiety and is capable ofbinding to the antigen being detected.

[0202] It will be understood in the art that the size of the subject andthe imaging system used will determine the quantity of imaging moietyneeded to produce diagnostic images. In the case of a radioisotopemoiety, for a human subject, the quantity of radioactivity injected willnormally range from about 5 to 20 millicuries of ^(99m)Tc. The labeledantibody will then preferentially accumulate at the location of cellswhich contain the specific protein. In vivo tumor imaging is describedin S. W. Burchiel et al., “Immunopharmacokinetics of RadiolabeledAntibodies and Their Fragments,” Chapter 13 in Tumor Imaging: TheRadiochemical Detection of Cancer, S. W. Burchiel and B. A. Rhodes,eds., Masson Publishing Inc. (1982).

[0203] Depending on several variables, including the type of label usedand the mode of administration, the time interval following theadministration for permitting the labeled molecule to preferentiallyconcentrate at sites in the subject and for unbound labeled molecule tobe cleared to background level is 6 to 48 hours or 6 to 24 hours or 6 to12 hours. In another embodiment the time interval followingadministration is 5 to 20 days or 5 to 10 days.

[0204] In one embodiment, monitoring of a disease, disorder or infectionis carried out by repeating the method for diagnosing the disease,disorder or infection, for example, one month after initial diagnosis,six months after initial diagnosis, one year after initial diagnosis,etc.

[0205] Presence of the labeled molecule can be detected in the subjectusing methods known in the art for in vivo scanning. These methodsdepend upon the type of label used. Skilled artisans will be able todetermine the appropriate method for detecting a particular label.Methods and devices that may be used in the diagnostic methods of theinvention include, but are not limited to, computed tomography (CT),whole body scan such as position emission tomography (PET), magneticresonance imaging (MRI), and sonography.

[0206] In a specific embodiment, the molecule is labeled with aradioisotope and is detected in the patient using a radiation responsivesurgical instrument (Thurston et al., U.S. Pat. No. 5,441,050). Inanother embodiment, the molecule is labeled with a fluorescent compoundand is detected in the patient using a fluorescence responsive scanninginstrument. In another embodiment, the molecule is labeled with apositron emitting metal and is detected in the patient using positronemission-tomography. In yet another embodiment, the molecule is labeledwith a paramagnetic label and is detected in a patient using magneticresonance imaging (MRI).

5.10. KITS

[0207] The invention also provides a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the pharmaceutical compositions of the invention.Optionally associated with such container(s) can be a notice in the formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals or biological products, which notice reflectsapproval by the agency of manufacture, use or sale for humanadministration.

[0208] The present invention provides kits that can be used in the abovemethods. In one embodiment, a kit comprises an antibody, fusion protein,or conjugated molecule, of the invention, preferably in a purified form,in one or more containers. In a specific embodiment, the kits of thepresent invention contain a substantially isolated antigen as a control.Preferably, the kits of the present invention further comprise a controlantibody, fusion protein, or conjugated molecule which does not reactwith the antigen included in the kit. In another specific embodiment,the kits of the present invention contain a means for detecting thebinding of an antibody, fusion protein, or conjugated molecule, to anantigen (e.g., the antibody, fusion protein, or conjugated molecule, maybe conjugated to a detectable substrate such as a fluorescent compound,an enzymatic substrate, a radioactive compound or a luminescentcompound, or a second antibody which recognizes the first antibody maybe conjugated to a detectable substrate). In specific embodiments, thekit may include a recombinantly produced or chemically synthesizedantigen. The antigen provided in the kit may also be attached to a solidsupport. In a more specific embodiment the detecting means of theabove-described kit includes a solid support to which antigen isattached. Such a kit may also include a non-attached reporter-labeledanti-human antibody. In this embodiment, binding of the antibody to theantigen can be detected by binding of the said reporter-labeledantibody.

5.11 IN VITRO AND IN VIVO ASSAYS FOR EXTENDED HALF-LIFE OF MODIFIED IGGHINGE-FC FRAGMENTS

[0209] The binding ability of modified IgGs and molecules comprising anIgG constant domain of FcRn fragment thereof to FcRn can becharacterized by various in vitro assays. PCT publication WO 97/34631 byWard discloses various methods in detail and is incorporated herein inits entirety by reference.

[0210] For example, in order to compare the ability of the modified IgGor fragments thereof to bind to FcRn with that of the wild type IgG, themodified IgG or fragments thereof and the wild type IgG can beradio-labeled and reacted with FcRn-expressing cells in vitro. Theradioactivity of the cell-bound fractions can be then counted andcompared. The cells expressing FcRn to be used for this assay arepreferably endothelial cell lines including mouse pulmonary capillaryendothelial cells (B10, D2.PCE) derived from lungs of B10.DBA/2 mice andSV40 transformed endothelial cells (SVEC) (Kim et al., J. Immunol.,40:457-465, 1994) derived from C3H/HeJ mice. However, other types ofcells, such as intestinal brush borders isolated from 10- to 14-day oldsuckling mice, which express sufficient number of FcRn can be also used.Alternatively, mammalian cells which express recombinant FcRn of aspecies of choice can be also utilized. After counting the radioactivityof the bound fraction of modified IgG or that of wild type, the boundmolecules can be then extracted with the detergent, and the percentrelease per unit number of cells can be calculated and compared.

[0211] Affinity of modified IgGs for FcRn can be measured by surfaceplasmon resonance (SPR) measurement using, for example, a BIAcore 2000(BIAcore Inc.) as described previously (Popov et al., Mol. Immunol.,33:493-502, 1996; Karlsson et al., J. Immunol. Methods, 145:229-240,1991, both of which are incorporated by reference in their entireties).In this method, FcRn molecules are coupled to a BIAcore sensor chip(e.g., CM5 chip by Pharmacia) and the binding of modified IgG to theimmobilized FcRn is measured at a certain flow rate to obtainsensorgrams using BIA evaluation 2.1 software, based on which on- andoff-rates of the modified IgG, constant domains, or fragments thereof,to FcRn can be calculated.

[0212] Relative affinities of modified IgGs or fragments thereof, andthe wild type IgG for FcRn can be also measured by a simple competitionbinding assay. Unlabeled modified IgG or wild type IgG is added indifferent amounts to the wells of a 96-well plate in which FcRn isimmobilize. A constant amount of radio-labeled wild type IgG is thenadded to each well. Percent radioactivity of the bound fraction isplotted against the amount of unlabeled modified IgG or wild type IgGand the relative affinity of the modified hinge-Fc can be calculatedfrom the slope of the curve.

[0213] Furthermore, affinities of modified IgGs or fragments thereof,and the wild type IgG for FcRn can be also measured by a saturationstudy and the Scatchard analysis.

[0214] Transfer of modified IgG or fragments thereof across the cell byFcRn can be measured by in vitro transfer assay using radiolabeled IgGor fragments thereof and FcRn-expressing cells and comparing theradioactivity of the one side of the cell monolayer with that of theother side. Alternatively, such transfer can be measured in vivo byfeeding 10- to 14-day old suckling mice with radiolabeled, modified IgGand periodically counting the radioactivity in blood samples whichindicates the transfer of the IgG through the intestine to thecirculation (or any other target tissue, e.g., the lungs). To test thedose-dependent inhibition of the IgG transfer through the gut, a mixtureof radiolabeled and unlabeled IgG at certain ratio is given to the miceand the radioactivity of the plasma can be periodically measured (Kim etal., Eur. J. Immunol., 24:2429-2434, 1994).

[0215] The half-life of modified IgG or fragments thereof can be measureby pharmacokinetic studies according to the method described by Kim etal. (Eur. J. of Immuno. 24:542, 1994), which is incorporated byreference herein in its entirety. According to this method, radiolabeledmodified IgG or fragments thereof is injected intravenously into miceand its plasma concentration is periodically measured as a function oftime, for example, at 3 minutes to 72 hours after the injection. Theclearance curve thus obtained should be biphasic, that is, α-phase andβ-phase. For the determination of the in vivo half-life of the modifiedIgGs or fragments thereof, the clearance rate in β-phase is calculatedand compared with that of the wild type IgG.

6. EXAMPLES

[0216] The following examples illustrate the production, isolation, andcharacterization of modified hinge-Fc fragments that have longer in vivohalf-lives.

6.1 LIBRARY CONSTRUCTION 6.1.1 REAGENTS

[0217] All chemicals were of analytical grade. Restriction enzymes andDNA-modifying enzymes were purchased from New England Biolabs, Inc.(Beverly, Mass.). Oligonucleotides were synthesized by MWG Biotech, Inc.(High Point, N.C.). pCANTAB5E phagemid vector, anti-E-tag-horseradishperoxydase conjugate, TG1 E. Coli strain, IgG Sepharose 6 Fast Flow andHiTrap protein A columns were purchased from APBiotech, Inc.(Piscataway, N.J.). VCSM13 helper phage and the Quick change mutagenesiskit were obtained from Stratagene (La Jolla, Calif.). CJ236 E. colistrain was purchased from Bio-Rad (Richmond, Calif.). BCA Protein AssayReagent Kit was obtained from Pierce (Rockford, Ill.). Lipofectamine2000 was purchased from Invitrogen, Inc. (Carlsbad, Calif.).

6.1.2 EXPRESSION AND PURIFICATION OF MURINE AND HUMAN FCRN

[0218] The amino acid sequences of human and mouse FeRn are SEQ ID NOs.84 and 85, respectively (see also Firan et al., Intern. Immunol.,13:993-1002, 2001 and Popov et al., Mol. Immunol., 33:521-530, 1996,both of which are incorporated herein by reference in their entireties).Human FcRn was also obtained following isolation from human placentaCDNA (Clontech, Palo Alto, Calif.) of the genes for humanβ2-microglobulin (Kabat et al., 1991, Sequences of Proteins ofImmunological Interest, U.S. Public Health Service, National Institutesof Health, Washington, D.C.) and codons −23 to 267 of the human α chain(Story et al., J. Exp. Med., 180:2377-2381, 1994) using standard PCRprotocols. Light and heavy chains along with their native signalsequence (Kabat et al., 1991, supra; Story et al., supra) were cloned inpFastBac DUAL and pFastBac I bacmids, respectively, and viral stocksproduced in Spodoptera frugiperda cells (Sf9) according to themanufacturer's instructions (Invitrogen, Carlsbad, Calif.). High-Fivecells were infected at a multiplicity of infection of 3 with thebaculoviruses encoding α and β2 chains using commercially availableprotocols (Invitrogen). Recombinant human FcRn was purified as follows:supernatant of infected insect cells was dialyzed into 50 mM MES(2-N-[Morpholino]ethansulfonic acid) pH 6.0 and applied to a 10 ml humanIgG Sepharose 6 Fast Flow column (APBiotech, Piscataway, N.J.). Resinwas washed with 200 ml 50 mM MES pH 6.0 and FcRn eluted with 0.1 MTris-Cl pH 8.0. Purified FcRn was dialyzed against 50 mM MES pH 6.0,flash frozen and stored at −70° C. The purity of proteins was checked bySDS-PAGE and HPLC.

6.1.3 PREPARATION OF TAA-CONTAINING ssDNA URACIL TEMPLATE

[0219] Construction of the libraries was based on a site directedmutagenesis strategy derived from the Kunkel method (Kunkel et al.,Methods Enzymol. 154:367-382, 1987). A human hinge-Fc gene spanningamino acid residues 226-478 (Kabat numbering, Kabat et al., 1991, supra)derived from MEDI-493 human IgG1 (Johnson et al., J. Infect. Disease,176:1215-1224, 1997), was cloned into the pCANTAB5E phagemid vector asan SfiI/NotI fragment. Four libraries were generated by introducingrandom mutations at positions 251, 252, 254, 255, 256 (library 1), 308,309, 311, 312, 314 (library 2), 385, 386, 387, 389 (library 3) and 428,433, 434, 436 (library 4). Briefly, four distinct hinge-Fc templateswere generated using PCR by overlap extension (Ho et al., Gene,15:51-59, 1989), each containing one TAA stop codon at position 252(library 1), 310 (library 2), 384 (library 3) or 429 (library 4), sothat only mutagenized phagemids will give rise to Fc-displaying phage.

[0220] Each TAA-containing single-stranded DNA (TAAssDNA) was thenprepared as follows: a single CJ236 E. coli colony harboring one of thefour relevant TAA-containing phagemids was grown in 10 ml 2× YT mediumsupplemented with 10 μg/ml chloramphenicol and 100 μg/ml ampicillin. AtOD₆₀₀=1, VCSM 13 helper phage was added to a final concentration of 10¹⁰pfu/ml. After 2 hours, the culture was transferred to 500 ml of 2× YTmedium supplemented with 0.25 μg/ml uridine, 10 μg/ml chloramphenicol,30 μg/ml kanamycin, 100 μg/ml ampicillin and grown overnight at 37° C.Phage were precipitated with PEG6000 using standard protocols (Sambrooket al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring HarborPress, Cold Spring Harbor, N.Y., Vols. 1-3) and purified using theQiaprep Spin M13 Kit (Qiagen, Valencia, Calif.) according to themanufacturer's instructions. 10 to 30 μg of each uracil-containingTAAssDNA template was then combined with 0.6 μg of the followingphosphorylated oligonucleotides (randomized regions underlined) in 50 mMTris-HCl, 10 mM MgCl₂, pH 7.5 in a final volume of 250 μl:

[0221] Library 1: (SEQ ID NO:120)5′-CATGTGACCTCAGGSNNSNNSNNGATSNNSNNGGTGTCCTTGGGTTT TGGGGGG-3′

[0222] Library 2: (SEQ ID NO:121)5′-GCACTTGTACTCCTTGCCATTSNNCCASNNSNNGTGSNNSNNGGTG AGGACGC-3′

[0223] Library 3:

[0224] 5′-GGTCTTGTAGTTSNNCTCSNNSNNSNNATTGCTCTCCC-3′ (SEQ ID NO:122)

[0225] Library 4: (SEQ ID NO:123)5′-GGCTCTTCTGCGTSNNGTGSNNSNNCAGAGCCTCATGSNNCACGGAG CATGAG-3′

[0226] where N=A, C, T or G and S=G or C.

6.1.4 SYNTHESIS OF HETERODUPLEX DNA

[0227] Appropriate, degenerate oligonucleotides were phosphorylated inthe presence of T4 polynucleotide kinase using the standard protocol.Ten to 30 μg of ssDNA U template and 0.6 μg of phosphorylatedoligonucleotide were combined in 50 mM Tris-HCl containing 10 mM MgCl₂,pH 7.5, to a final volume of 250 μl and incubated at 90° C. for 2minutes, 50° C. for 3 minutes, and 20° C. for 5 minutes. Synthesis ofthe heteroduplex DNA was carried out by adding 30 units of both T4 DNAligase and T7 DNA polymerase in the presence of 0.4 mM ATP, 1 mM dNTPsand 6 mM DTT and the mixture was incubated for 4 hours at 20° C. Theheteroduplex DNA thus produced was then purified and desalted usingQiagen Qiaquick DNA purification Kit (Qiagen, CA).

6.1.5 ELECTROPORATION

[0228] 300 μl electrocompetent TG1 E. coli cells were electroporatedwith 1 to 5 μg of the heteroduplex DNA in a 2.5 kV field using 200Ω and25 μF capacitance until a library size of 1×10⁸ (library 1 and 2) or1×10⁷ (library 3 and 4) was reached. The cells were resuspended in 2 mlSOC medium and the procedure was repeated 6 to 10 times. The diversitywas assessed by titration of recombinant E. coli. The pulsed cells wereincubated in 50 ml SOC medium for 30 minutes at 37° C. under agitation,centrifuged, and resuspended in 500 ml 2× YT containing 100 μg/mlampicillin and 10¹⁰ pfu/ml of VCSM13 helper phage. The culture wasincubated overnight at 37° C. and the cells were pelleted bycentrifugation. The phage in the supernatant which express mutatedhinge-Fc portion on its GIII-coat protein were precipitated with PEG6000as previously described (Sambrook et al., 1989, supra) and resuspendedin 5 ml of 20 mM MES, pH 6.0.

6.2 PANNING OF THE LIBRARY

[0229] Phage were panned using an ELISA-based approach. A 96-well ELISAplate was coated with 100 μl/well of 0.01 mg/ml murine FcRn in sodiumcarbonate buffer, pH 9.0, at 4° C. overnight and then blocked with 4%skimmed milk at 37° C. for 2 hours. In each well of the coated plate,100-150 μl of the phage suspension (about 10¹³ phage in total) in 20 mMMES, pH 6.0, containing 5% milk and 0.05% Tween 20, were placed andincubated at 37° C. for two to three hours with agitation.

[0230] After the incubation, the wells were washed with 20 mM MES, pH6.0, containing 0.2% Tween 20 and 0.3 M NaCl about thirty times at roomtemperature. The bound phage were eluted with 100 μl/well of PBS, pH7.4, at 37° C. for 30 minutes.

[0231] The eluted phage were then added to the culture of exponentiallygrowing E. coli cells and propagation was carried out overnight at 37°C. in 250 ml 2× YT supplemented with 100 μg/ml ampicillin and 10¹⁰pfu/ml of VCSM13 helper phage. Propagated phage were collected bycentrifugation followed by precipitation with PEG and the panningprocess was repeated up to a total of six times.

[0232] For the phage library containing mutations in residues 308-314(H310 and W313 fixed), the phage expressing hinge-Fc region with higheraffinities for FcRn were enriched by each panning process as shown inTable IV. The panning results of the library for the mutations in theresidues 251-256 (I253 fixed) and that of the library for the mutationsin the residues 428-436 (H429, E430, A431, L432, and H435 fixed), areshown in Tables V and VI, respectively. Furthermore, the panning resultsof the library for the mutations in the residues 385-389 (E388 fixed) isshown in Table VII. TABLE IV PANNING OF LIBRARY (RESIDUES 308-314; H310AND W313 FIXED) pCANTAB5E-KUNKEL-muFcRn (MURINE FcRn) OUTPUT ENRICHMENTPANNING + FcRn − FcRn RATIO 1st Round 1.1 × 10⁵   0.5 × 10⁵  2 2nd Round1 × 10⁴ 0.2 × 10⁴  5 3rd Round 9 × 10⁴ 0.3 × 10⁴ 30 4th Round 3 × 10⁵  2 × 10⁴ 15

[0233] TABLE V PANNING OF LIBRARY (RESIDUES 251-256; I253 FIXED)pCANTAB5E-KUNKEL-muFcRn OUTPUT ENRICHMENT PANNING + FcRn − FcRn RATIO1st Round 2.5 × 10⁵ 1 × 10⁵ 2.5 2nd Round   6 × 10⁴ 2 × 10⁴ 3.0 3rdRound   8 × 10⁵ 4 × 10⁴ 20 4th Round 1.2 × 10⁶ 5 × 10⁴ 24 5th Round 3.0× 10⁶ 6 × 10⁴ 50

[0234] TABLE VI PANNING OF LIBRARY (RESIDUES 428-436; H429, E430, A431,L432, AND H435 FIXED) pCANTAB5E-KUNKEL-muFcRn OUTPUT ENRICHMENTPANNING + FcRn − FcRn RATIO 1st Round 2.3 × 10⁵   0.9 × 10⁵   2.5 2ndRound 3 × 10⁴ 1 × 10⁴ 3 3rd Round 2 × 10⁵ 2 × 10⁴ 10 4th Round 8 × 10⁵ 5× 10⁴ 16

[0235] TABLE VII PANNING OF LIBRARY (RESIDUES 385-389; E388 FIXED)pCANTAB5E-KUNKEL-muFcRn OUTPUT ENRICHMENT PANNING + FcRn − FcRn RATIO1st Round 4.2 × 10⁵ 3.8 × 10⁵   1.1 2nd Round   5 × 10⁴ 0.3 × 10⁴   173rd Round 3.5 × 10⁵ 1 × 10⁴ 35 4th Round 5.5 × 10⁵ 4 × 10⁴ 14 5th Round7.5 × 10⁵ 5 × 10⁴ 15 6th Round   2 × 10⁶ 1 × 10⁵ 20

6.3 IDENTIFICATION OF ISOLATED CLONES FROM PANNING

[0236] After each panning process, phage were isolated and the nucleicacids encoding the expressed peptides which bound to FcRn were sequencedby a standard sequencing method such as by dideoxynucleotide sequencing(Sanger et al., Proc. Natl. Acad. Sci USA, 74:5463-5467, 1977) using aABI3000 genomic analyzer (Applied Biosystems, Foster City, Calif.).

[0237] As a result of panning, two mutants were isolated from the phagelibrary containing mutations in residues 308-314 (H310 and W313 fixed),thirteen mutants from the library for residues 251-256 (I253 fixed), sixmutants from the library for residues 428-436 (H429, E430, A431, L432,and H435 fixed), and nine mutants from the library for residues 385-389(E388 fixed). The mutants isolated from the libraries are listed inTable VIII. TABLE VIII MUTANTS ISOLATED BY PANNING LIBRARY MUTANTS*251-256 Leu Tyr Ile Thr Arg Glu (SEQ ID NO:90) Leu

Tyr Ile Ser Arg Thr (SEQ ID NO:91) Leu

Tyr Ile Ser Arg

Ser (SEQ ID NO:92) Leu

Tyr Ile Ser Arg

Arg (SEQ ID NO:93) Leu

Tyr Ile Ser Arg

Gln (SEQ ID NO:94) Leu

Trp Ile Ser Arg Thr (SEQ ID NO:95) Leu Tyr Ile Ser Leu Gln (SEQ IDNO:96) Leu Phe Ile Ser Arg Asp (SEQ ID NO:97) Leu Phe Ile Ser Arg Thr(SEQ ID NO:98) Leu Phe Ile Ser Arg Arg (SEQ ID NO:99) Leu Phe Ile ThrGly Ala (SEQ ID NO:100) Leu Ser Ile Ser Arg Glu (SEQ ID NO:101) Arg ThrIle Ser Ile Ser (SEQ ID NO:102) 308-314 Thr Pro His Ser Asp Trp Leu (SEQID NO:103) Ile Pro His Glu Asp Trp Leu (SEQ ID NO:104) 385-389Arg Thr Arg Glu Pro (SEQ ID NO:105)

Asp

Pro Pro Gln

Ser (SEQ ID NO:106) Ser Asp Pro Glu Pro (SEQ ID NO:107) Thr Ser His GluAsn (SEQ ID NO:108) Ser Lys Ser Glu Asn (SEQ ID NO:109) His Arg Ser GluAsn (SEQ ID NO:110) Lys Ile Arg Glu Asn (SEQ ID NO:111) Gly Ile Thr GluSer (SEQ ID NO:112) Ser Met Ala Glu Pro (SEQ ID NO:113) 428-436 Met HisGln Ala Leu

Arg

Tyr His

His (SEQ ID NO:114) Met His Glu Ala Leu His Phe His His (SEQ ID NO:115)Met His Glu Ala Leu Lys Phe His His (SEQ ID NO:116) Met His Glu Ala LeuSer Tyr His Arg (SEQ ID NO:117) Thr His Glu Ala Leu His Tyr His Thr (SEQID NO:118) Met His Glu Ala Leu His Tyr His Tyr (SEQ ID NO:119)

[0238] The underlined sequences in Table VIII correspond to sequencesthat occurred 10 to 20 times in the final round of panning and thesequences in italics correspond to sequences that occurred 2 to 5 timesin the final round of panning. Those sequences that are neitherunderlined nor italicized occurred once in the final round of panning.

6.4 EXPRESSION AND PURIFICATION OF SOLUBLE MUTANT HINGE-FC REGION

[0239] The genes encoding mutated hinge-Fc fragments are excised withappropriate restriction enzymes and recloned into an expression vector,for example, VβpelBhis (Ward, J. Mol. Biol., 224:885-890, 1992). Vectorscontaining any other type of tag sequence, such as c-myc tag,decapeptide tag (Huse et al., Science, 246:1275-1281, 1989), Flag™(Immunex) tags, can be used. Recombinant clones, such as E. coli, aregrown and induced to express soluble hinge-Fc fragments, which can beisolated from the culture media or cell lysate after osmotic shock,based on the tag used, or by any other purification methods well knownto those skilled in the art and characterized by the methods as listedbelow.

6.5 CONSTRUCTION, PRODUCTION AND PURIFICATION OF IgG1 VARIANTS

[0240] Representative Fc mutations such as I253A, M252Y/S254T/T256E,M252W, M252Y, M252Y/T256Q, M252F/T256D, V308T/L309P/Q311S,G385D/Q386P/N389S, G385R/Q386T/P387R/N389P, H433K/N434F/Y436H, andN434F/Y436 were incorporated into the human IgG1 MEDI-493 (SYNAGIS®)(Johnson et al., 1997, supra). The heavy chain was subjected tosite-directed mutagenesis using a Quick Change Mutagenesis kit(Stratagene, La Jolla, Calif.) according to the manufacturer'sinstructions and sequences were verified by didoxynucleotide sequencingusing a ABI3000 (Applied Biosystems, Foster City, Calif.) sequencer. Thedifferent constructions were expressed transiently in human embryonickidney 293 cells using a CMV immediate-early promoter and dicistronicoperon in which IgG1/V_(H) is cosecreted with IgG1/V_(L) (Johnson etal., 1997, supra). Transfection was carried out using Lipofectamine 2000(Invitrogen, Carlsbad, Calif.) and standard protocols. IgGs werepurified from the conditioned media directly on 1 ml HiTrap protein Acolumns according to the manufacterer's instructions (APBiotech).

6.6 CHARACTERIZATION OF MUTATED HINGE-FC REGION 6.6.1 IN VITROCHARACTERIZATION HPLC AND SDS-PAGE

[0241] Following the purification, general characteristics such asmolecular weight and bonding characteristics of the modified hinge-Fcfragments may be studied by various methods well known to those skilledin the art, including SDS-PAGE and HPLC.

[0242] FcRn Binding Assay

[0243] Binding activity of modified hinge-Fc fragments can be measuredby incubating radio-labeled wild-type hinge-Fc or modified hinge-Fc withthe cells expressing either mouse or human FcRn. Typically, endothelialcell lines such as SV40 transformed endothelial cells (SVEC) (Kim etal., J. Immunol., 40:457-465, 1994) are used. After incubation with thehinge-Fc fragments at 37° C. for 16-18 hours, the cells are washed withmedium and then detached by incubation with 5 mM Na₂EDTA in 50 mMphosphate buffer, pH 7.5, for 5 minutes. The radioactivity per 10⁷ cellsis measured.

[0244] Then, the cells are resuspended in 2 ml of 2.5 mg/ml CHAPS, 0.1 MTris-HCl pH 8.0 containing 0.3 mg/ml PMSF, 25 mg/ml pepstatin and 0.1mg/ml aprotinin and incubated for 30 minutes at room temperature. Thecell suspension is then centrifuged and the supernatant separated. Theradioactivity of the supernatant is measured and used to calculate theamount of the hinge-Fc fragments extracted per 10⁷ cells.

[0245] The K_(d) for the interaction of wild type human IgG1 with murineand human FcRn (269 and 2527 nM, respectively) agree well with thevalues determined by others (265 and 2350 nM, respectively, Firan etal., 2001, supra). The I253A mutation virtually abolishes binding tohuman and murine FcRn, as reported by others (Kim et al., Eur. J.Immunol., 29:2819-2825, 1991; Shields et al., J. Biol. Chem.,276:6591-6604, 2001). This is not the result of misfolding of theantibody as this mutant retains the same specific activity than the wildtype molecule (SYNAGIS®) in a microneutralization assay (Johnson et al.,1997, supra; data not shown).

[0246] Human IgG1 mutants with increased binding affinity towards bothmurine and human FcRn were generated (Table VIII). Improvements incomplex stability were overall less marked for the human IgG1-human FcRnpair than for the human IgG1-murine FcRn compared to wild type IgG1 were30-(ΔΔG=2.0 kcal/mol for N434F/Y436H) and 11-(ΔΔG=1.4 kcal/mol forM252Y/S254Y/S254T/T256E) fold, respectively. However, ranking of themost critical positions remain unchanged when comparing human and murineFcRn: the largest increases in IgG1-murine FcRn complex stability(ΔΔG>1.3 kcal/mol) occurred on mutations at positions 252, 254, 256(M252Y/S254T/T256E and M252W) and 433, 434, 436 (H433K/N434F/Y436H andN434F/Y436H). Likewise, the same mutations were found to have the mostprofound impact on the IgG1-human FcRn interaction and also resulted inthe largest increases in complex stability (ΔΔG>1.0 kcal/mol).Substitutions at positions 308, 309, 311, 385, 386, 387 and 389 hadlittle or no effect on the stability of the complexes involving human ormurine FcRn (ΔΔG<0.5 kcal/mol). Residues at the center of the Fc-FcRncombining site contribute significantly more to improvement in complexstability than residues at the periphery (FIG. 9).

[0247] Efficient binding of human Fc to murine FcRn apparently requiresthe presence of several wild type Fc residues. For example, leucine isvery conserved at 251, arginine at 255, aspartic acid at 310, leucine at314 and methionine at 428 (FIG. 6). Another specificity trend isobserved when one considers positions 308, 309, and 311 where threonine,proline, and serine, respectively, are very strongly favored over thecorresponding wild type residues (FIG. 6). However, generation of thisstrong consensus sequences does not correlate with the magnitude ofincrease in affinity as V308T/L309P/Q311S binds less than 2-fold betterthan the wild type IgG1 to both human and murine FcRn (Table IX).

[0248] Increases in affinity can be strongly dependent upon residuesubstitution at one ‘hot spot’ position. For example, the singlemutation M252Y causes an increase in binding to murine FcRn by 9-fold,whereas additional mutations bring little (M252Y/S254T/T256E) or no(M252Y/T256Q) added benefit. The same trend is observed for the humanreceptor, although to a lesser extent. Indeed, M252Y/S254T/T256E shows amarked improvement of 2.5-fold in affinity compared to M252Y. Thisprobably reflects the differences between the binding site of human andmurine FcRn (West and Bjorkman, Biochemistry, 39:9698-9708, 2000).

[0249] Phage-derived IgG1 mutants exhibiting a significant increase inaffinity towards murine FcRn (ΔΔG>1.3 kcal/mol) also showed significantbinding activity to the receptor at pH 7.2 when compared to wild typeIgG1 (FIGS. 8A-H). IgG1 mutants with moderate increase in affinity(ΔΔG<0.3 kcal/mol) bound very poorly at pH 7.2 (data not shown). Incontrast, IgG1 mutants with large (ΔΔG>1.0 kcal/mol) increase inaffinity towards human FcRn exhibited only minimal binding at pH 7.4when compared to wild type IgG1 (FIGS. 8A-H). TABLE IX DISSOCIATIONCONSTANTS AND RELATIVE FREE ENERGY CHANGES FOR THE BINDING OF IgG1/FCMUTANTS TO MURINE AND HUMAN FcRn* Dissociation Dissociation ConstantConstant Fc/Murine ΔΔG Fe/Human ΔΔG MUTANT FcRn (nM) (kcal/mol) FcRn(mM) (kcal/mol) wild type 269 ± 1  2527 ± 117 I253A NB NA NB NAM252Y/S254T/ 27 ± 6 1.4 225 ± 10 1.4 T256E M252W 30 ± 1 1.3 408 ± 24 1.1M252Y 41 ± 7 1.1 532 ± 37 0.9 M252Y/T256Q 39 ± 8 1.1  560 ± 102 0.9M252F/T256D 52 ± 9 1.0  933 ± 170 0.6 V308T/L309P/ 153 ± 23 0.3 1964 ±84  0.1 Q311S G385D/Q386P/ 187 ± 10 0.2 2164 ± 331 0.1 N389SG385R/Q386T/ 147 ± 24 0.4 1620 ± 61  0.3 P387R/N389P H433K/N434F/ 14 ± 21.8 399 ± 47 1.1 Y436H N434F/Y436H  9 ± 1 2.0 493 ± 7  1.0

[0250] FcRn-Mediated Transfer Assay

[0251] This assay follows the protocol disclosed in PCT publication WO97/34631. Radiolabeled modified hinge-Fc fragments at variousconcentration (1 μg/ml-1 mg/ml) are added to the one side of thetranswell and the transfer of the fragments mediated by FcRn-expressingmonolayer of the cells can be quantitated by measuring the radioactivityon the other side of the transwell.

6.6.2 IN VIVO PHARMACOKINETIC STUDY

[0252] In order to determine the half-life of the modified IgG hinge-Fc,modified hinge-Fc fragments are radiolabelled with ¹²⁵I (approximatespecific activity of 10⁷ cpm/μg) and dissolved in saline (pH 7.2). Thesolution is injected intravenously into BALB/c mice (Harlan,Indianapolis, Ind.), which have been given NaI-containing waterpreviously to block the thyroid, in a volume not more than 150 μl andwith a radioactivity of 10×10⁶-50×10⁶ cpm. The mice are bled from theretro-orbital sinus at various time points, for example, at 3 minutes to72 hours after the injection, into heparinized capillary tubes and theplasma collected from each sample is counted for radioactivity.

[0253] To generate the data provided in FIG. 10, 10 animals were usedfor each molecule assayed with 2.5 μg of antibody injected per animal.Antibody serum levels were determined using an anti-human IgG ELISA(FIG. 10). There seems to be an inverse correlation between affinity tomouse FcRn and persistence in serum. This might be due to thesignificant amount of binding of the mutants observed at pH 7.2, whichleads to the sequestration (i.e., lack of release in the serum) of themolecules. Preliminary data (not shown) suggests increased transport ofthe mutants to the lung. Additionally, since the mutants exhibit lowerlevels of binding to human FcRn than murine FcRn (see FIGS. 8A-H),antibody serum levels are expected to be higher in primates and humans.

6.6.3 SURFACE PLASMON RESONANCE ANALYSES

[0254] The interaction of soluble murine and human FcRn with immobilizedhuman IgG1 variants was monitored by surface plasmon resonance detectionusing a BIAcore 3000 instrument (Pharmacia Biosensor, Uppsala, Sweden).No aggregated material which could interfere with affinity measurements(van der Merwe et al., EMBO J., 12:4945-4954, 1993; van der Merwe etal., Biochemistry, 33:10149-10160, 1994) was detected by gel filtration.Protein concentrations were calculated by the bicinchoninic acid (BCA)method for both human and murine FcRn or using the 1% extinctioncoefficient at 280 nm of 1.5 for IgG1 wild type and variants. The latterwere coupled to the dextran matrix of a CM5 sensor chip (PharmaciaBiosensor) using an Amine Coupling Kit as described (Johnson et al.,supra). The protein concentrations ranged from 3-5 μg/ml in 10 mM sodiumacetate, pH 5.0. The activation period was set for 7 minutes at a flowrate of 10 μl/min and the immobilization period was set to between 10and 20 minutes at a flow rate of 10 μl/min. Excess reactive esters werequenched by injection of 70 μl of 1.0 methanolamine hydrochloride, pH8.5. This typically resulted in the immobilization of between 500 and4000 resonance units (RU). Human and murine FcRn were buffer exchangedagainst 50 mM PBS buffer pH 6.0 containing 0.05% Tween 20. Dilutionswere made in the same buffer. All binding experiments were performed at25° C. with concentrations ranging from 120 to 1 μg/ml at a flow rate of5 to 10 μl/min; data were collected for 25 to 50 minutes and three1-minute pulses of PBS buffer pH 7.2 were used to regenerate thesurfaces. FcRn was also flowed over an uncoated cell and the sensorgramsfrom these blank runs subtracted from those obtained with IgG1-coupledchips. Runs were analyzed using the software BIAevaluation 3.1(Pharmacia). Association constants (K_(A)S) were determined fromScatchard analysis by measuring the concentration of free reactants andcomplex at equilibrium after correction for nonspecific binding. Inequilibrium binding BIAcore experiments (Karlsson et al., 1991, supra;van der Merwe et al., 1993, supra; van der Merwe et al., 1994, supra;Raghavan et al., Immunity, 1:303-315, 1994; Malchiodi et al., J. Exp.Med., 182:1833-1845, 1995), the concentration of the complex can beassessed directly as the steady-state response. The concentration offree analyte (human or murine FcRn) is equal to the bulk analyteconcentration since analyte is constantly replenished during sampleinjection. The concentration of free ligand on the surface of the sensorchip can be derived from the concentration of the complex and from thetotal binding capacity of the surface as K_(A)=R_(eq)/C(R_(max)−R_(eq))where C is the free analyte concentration, R_(eq) is the steady-stateresponse, and R_(max) is the total surface binding capacity.Rearranging, the equation reads: R_(eq)/C=K_(A) R_(max)−K_(A) R_(eq).

[0255] A plot of R_(eq)/C versus R_(eq) at different analyteconcentrations thus gives a straight line from which K_(A) can becalculated (see Table IX). Errors were estimated as the standarddeviation for two or three independent determinations and were <20%.

[0256] Representative mutations identified after panning libraries 1through 4 (FIG. 6, Table VIII) were introduced into the Fc portion of ahuman IgG1. Injection of different concentrations of human or murineFcRn over the immobilized IgG1 variants gave concentration-dependentbinding. Typical resonance profiles for equilibrium binding of themutant M252Y/S254T/T256E to murine and human FcRn are shown in FIGS. 7Aand B. To estimate apparent K_(A)s, concentrations of FcRn ranging from120 to 1 μg/ml were used. In all cases, equilibrium (ornear-equilibrium) binding levels were reached within 50 minutes. Toestimate the increase in RU resulting from the non specific effect ofprotein on the bulk refractive index, binding of FcRn to an uncoatedcell was measured and the sensorgrams from these blank runs subtractedfrom those obtained with IgG1-coupled chips. The scatchard plots for thebinding of the mutant M252Y/S254T/T256E to murine and human FcRn areshown in FIGS. 7C and D. The plots were all linear, and apparent K_(A)Swere calculated from the relevant slopes. Measurements were carried outin duplicate or triplicate and confirmed that the immobilized IgGsretained their original binding activity.

[0257] Since there are two non-equivalent binding sites on mouse IgG1for murine FcRn with affinities of <130 nM and 6 μM (Sanchez et al.,Biochemistry, 38:9471-9476, 1999; Schuck et al., Mol. Immunol.,36:1117-1125, 1999; Ghetie and Ward, Ann. Rev. Immunol., 18:739-766,2000), the receptor was used in solution to avoid avidity effects thatarise when IgG1 binds to immobilized FcRn. Consistent with this,systematically higher affinities are observed when FcRn, rather thanIgG, immobilized on the biosensor chip (Popov et al., 1996, supra;Vaughn and Bjorkman, Biochemistry, 36:9374-9380, 1997; Martin andBjorkman, Biochemistry, 38:12639-12647; West and Bjorkman, Biochemistry,39:9698-9708, 2000). Under our experimental BIAcore conditions, mainlyinteractions corresponding to the higher-affinity association (i.e.single liganded-recptor) are measured, according for the linearity ofthe scatchard plots (FIGS. 7C and D).

[0258] BIAcore analysis was also used to compare the affinity of wildtype IgG1 and IgG1 mutants. Phage-derived IgG1 mutants exhibiting asignificant increase in affinity towards murine FcRn at pH 6.0 (ΔΔG≧1.0kcal/mol) also shoed significant binding to the mouse receptor at pH 7.2with SPR signal_(pH7 4)/SPR signal_(pH6 0)>0.6 at saturation. IgG1mutants with moderate increase in affinity towards murine FcRn at pH 6.0(ΔΔG<0.4 kcal/mol) bound very poorly to the mouse receptor at pH 7.2. Incontrast, IgG1 mutants exhibiting large affinity increase towards humanFcRn at pH 6.0 (ΔΔG≧1.0 kcal/mol) only showed minimal binding to thehuman receptor at pH 7.4 with SPR signal_(pH7 4)/SPR signal_(pH6 0)<0.15at saturation.

[0259] Those skilled in the art will recognize, or be able to ascertainusing no more routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

[0260] All publications, patents and patent applications mentioned inthis specification are herein incorporated by reference into thespecification to the same extent as if each individual publication,patent or patent application was specifically and individually indicatedto be incorporated herein by reference.

1 118 1 7 PRT Homo sapiens 1 Thr Ser Gly Met Ser Val Gly 1 5 2 16 PRTHomo sapiens 2 Asp Ile Trp Trp Asp Asp Lys Lys Asp Tyr Asn Pro Ser LeuLys Ser 1 5 10 15 3 10 PRT Homo sapiens 3 Ser Met Ile Thr Asn Trp TyrPhe Asp Val 1 5 10 4 10 PRT Homo sapiens 4 Lys Cys Gln Leu Ser Val GlyTyr Met His 1 5 10 5 7 PRT Homo sapiens 5 Asp Thr Ser Lys Leu Ala Ser 15 6 9 PRT Homo sapiens 6 Phe Gln Gly Ser Gly Tyr Pro Phe Thr 1 5 7 120PRT Homo sapiens 7 Gln Val Thr Leu Arg Glu Ser Gly Pro Ala Leu Val LysPro Thr Gln 1 5 10 15 Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe SerLeu Ser Thr Ser 20 25 30 Gly Met Ser Val Gly Trp Ile Arg Gln Pro Pro GlyLys Ala Leu Glu 35 40 45 Trp Leu Ala Asp Ile Trp Trp Asp Asp Lys Lys AspTyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr SerLys Asn Gln Val 65 70 75 80 Val Leu Lys Val Thr Asn Met Asp Pro Ala AspThr Ala Thr Tyr Tyr 85 90 95 Cys Ala Arg Ser Met Ile Thr Asn Trp Tyr PheAsp Val Trp Gly Ala 100 105 110 Gly Thr Thr Val Thr Val Ser Ser 115 1208 106 PRT Homo sapiens misc_feature VL Domain 8 Asp Ile Gln Met Thr GlnSer Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr IleThr Cys Lys Cys Gln Leu Ser Val Gly Tyr Met 20 25 30 His Trp Tyr Gln GlnLys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35 40 45 Asp Thr Ser Lys LeuAla Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr GluPhe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp 65 70 75 80 Asp Phe Ala ThrTyr Tyr Cys Phe Gln Gly Ser Gly Tyr Pro Phe Thr 85 90 95 Phe Gly Gly GlyThr Lys Leu Glu Ile Lys 100 105 9 120 PRT Homo sapiens misc_feature VHDomain 9 Gln Val Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln1 5 10 15 Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser ThrAla 20 25 30 Gly Met Ser Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala LeuGlu 35 40 45 Trp Leu Ala Asp Ile Trp Trp Asp Asp Lys Lys Asp Tyr Asn ProSer 50 55 60 Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn GlnVal 65 70 75 80 Val Leu Lys Val Thr Asn Met Asp Pro Ala Asp Thr Ala ThrTyr Tyr 85 90 95 Cys Ala Arg Ser Met Ile Thr Asn Phe Tyr Phe Asp Val TrpGly Ala 100 105 110 Gly Thr Thr Val Thr Val Ser Ser 115 120 10 7 PRTHomo sapiens 10 Thr Ala Gly Met Ser Val Gly 1 5 11 106 PRT Homo sapiens11 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 510 15 Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Arg Val Gly Tyr Met 2025 30 His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 3540 45 Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 5055 60 Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp 6570 75 80 Asp Phe Ala Thr Tyr Tyr Cys Phe Gln Gly Ser Gly Tyr Pro Phe Thr85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 12 10 PRT Homosapiens 12 Ser Met Ile Thr Asn Phe Tyr Phe Asp Val 1 5 10 13 106 PRTHomo sapiens 13 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala SerVal Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser ValGly Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys LeuLeu Ile Tyr 35 40 45 Asp Thr Phe Lys Leu Ala Ser Gly Val Pro Ser Arg PheSer Gly Ser 50 55 60 Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser LeuGln Pro Asp 65 70 75 80 Asp Phe Ala Thr Tyr Tyr Cys Phe Gln Phe Ser GlyTyr Pro Phe Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 10514 10 PRT Homo sapiens 14 Ser Ala Ser Ser Ser Val Gly Tyr Met His 1 5 1015 7 PRT Homo sapiens 15 Asp Thr Phe Lys Leu Ala Ser 1 5 16 9 PRT Homosapiens 16 Phe Gln Phe Ser Gly Tyr Pro Phe Thr 1 5 17 120 PRT Homosapiens 17 Gln Val Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro ThrGln 1 5 10 15 Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu SerThr Pro 20 25 30 Gly Met Ser Val Gly Trp Ile Arg Gln Pro Pro Gly Lys AlaLeu Glu 35 40 45 Trp Leu Ala Asp Ile Trp Trp Asp Asp Lys Lys His Tyr AsnPro Ser 50 55 60 Leu Lys Asp Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys AsnGln Val 65 70 75 80 Val Leu Lys Val Thr Asn Met Asp Pro Ala Asp Thr AlaThr Tyr Tyr 85 90 95 Cys Ala Arg Asp Met Ile Phe Asn Phe Tyr Phe Asp ValTrp Gly Ala 100 105 110 Gly Thr Thr Val Thr Val Ser Ser 115 120 18 7 PRTHomo sapiens 18 Thr Pro Gly Met Ser Val Gly 1 5 19 16 PRT Homo sapiens19 Asp Ile Trp Trp Asp Asp Lys Lys His Tyr Asn Pro Ser Leu Lys Asp 1 510 15 20 10 PRT Homo sapiens 20 Asp Met Ile Phe Asn Phe Tyr Phe Asp Val1 5 10 21 106 PRT Homo sapiens misc_feature VL Domain 21 Asp Ile Gln MetThr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg ValThr Ile Thr Cys Ser Leu Ser Ser Arg Val Gly Tyr Met 20 25 30 His Trp TyrGln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35 40 45 Asp Thr PheTyr Leu Ser Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser GlyThr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp 65 70 75 80 Asp PheAla Thr Tyr Tyr Cys Phe Gln Gly Ser Gly Tyr Pro Phe Thr 85 90 95 Phe GlyGly Gly Thr Lys Val Glu Ile Lys 100 105 22 10 PRT Homo sapiens 22 SerLeu Ser Ser Arg Val Gly Tyr Met His 1 5 10 23 7 PRT Homo sapiens 23 AspThr Phe Tyr Leu Ser Ser 1 5 24 120 PRT Homo sapiens misc_feature VHDomain 24 Gln Val Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro ThrGln 1 5 10 15 Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu SerThr Pro 20 25 30 Gly Met Ser Val Gly Trp Ile Arg Gln Pro Pro Gly Lys AlaLeu Glu 35 40 45 Trp Leu Ala Asp Ile Trp Trp Asp Gly Lys Lys His Tyr AsnPro Ser 50 55 60 Leu Lys Asp Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys AsnGln Val 65 70 75 80 Val Leu Lys Val Thr Asn Met Asp Pro Ala Asp Thr AlaThr Tyr Tyr 85 90 95 Cys Ala Arg Asp Met Ile Phe Asn Phe Tyr Phe Asp ValTrp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser 115 120 25 16PRT Homo sapiens 25 Asp Ile Trp Trp Asp Gly Lys Lys His Tyr Asn Pro SerLeu Lys Asp 1 5 10 15 26 106 PRT Homo sapiens 26 Asp Ile Gln Met Thr GlnSer Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr IleThr Cys Ser Leu Ser Ser Arg Val Gly Tyr Met 20 25 30 His Trp Tyr Gln GlnLys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35 40 45 Asp Thr Arg Gly LeuPro Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr GluPhe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp 65 70 75 80 Asp Phe Ala ThrTyr Tyr Cys Phe Gln Gly Ser Gly Tyr Pro Phe Thr 85 90 95 Phe Gly Gly GlyThr Lys Val Glu Ile Lys 100 105 27 7 PRT Homo sapiens 27 Asp Thr Arg GlyLeu Pro Ser 1 5 28 120 PRT Homo sapiens 28 Gln Val Thr Leu Arg Glu SerGly Pro Ala Leu Val Lys Pro Thr Gln 1 5 10 15 Thr Leu Thr Leu Thr CysThr Phe Ser Gly Phe Ser Leu Ser Thr Pro 20 25 30 Gly Met Ser Val Gly TrpIle Arg Gln Pro Pro Gly Lys Ala Leu Glu 35 40 45 Trp Leu Ala Asp Ile TrpTrp Asp Gly Lys Lys His Tyr Asn Pro Ser 50 55 60 Leu Lys Asp Arg Leu ThrIle Ser Lys Asp Thr Ser Lys Asn Gln Val 65 70 75 80 Val Leu Lys Val ThrAsn Met Asp Pro Ala Asp Thr Ala Thr Tyr Tyr 85 90 95 Cys Ala Arg Asp MetIle Phe Asn Trp Tyr Phe Asp Val Trp Gly Gln 100 105 110 Gly Thr Thr ValThr Val Ser Ser 115 120 29 10 PRT Homo sapiens 29 Asp Met Ile Phe AsnTrp Tyr Phe Asp Val 1 5 10 30 106 PRT Homo sapiens 30 Asp Ile Gln MetThr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg ValThr Ile Thr Cys Ser Pro Ser Ser Arg Val Gly Tyr Met 20 25 30 His Trp TyrGln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35 40 45 Asp Thr MetArg Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser GlyThr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp 65 70 75 80 Asp PheAla Thr Tyr Tyr Cys Phe Gln Gly Ser Gly Tyr Pro Phe Thr 85 90 95 Phe GlyGly Gly Thr Lys Val Glu Ile Lys 100 105 31 10 PRT Homo sapiens 31 SerPro Ser Ser Arg Val Gly Tyr Met His 1 5 10 32 7 PRT Homo sapiens 32 AspThr Met Arg Leu Ala Ser 1 5 33 120 PRT Homo sapiens 33 Gln Val Thr LeuArg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln 1 5 10 15 Thr Leu ThrLeu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Pro 20 25 30 Gly Met SerVal Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu 35 40 45 Trp Leu AlaAsp Ile Trp Trp Asp Gly Lys Lys His Tyr Asn Pro Ser 50 55 60 Leu Lys AspArg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val 65 70 75 80 Val LeuLys Val Thr Asn Met Asp Pro Ala Asp Thr Ala Thr Tyr Tyr 85 90 95 Cys AlaArg Asp Met Ile Phe Asn Trp Tyr Phe Asp Val Trp Gly Gln 100 105 110 GlyThr Thr Val Thr Val Ser Ser 115 120 34 106 PRT Homo sapiens 34 Asp IleGln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 AspArg Val Thr Ile Thr Cys Ser Leu Ser Ser Arg Val Gly Tyr Met 20 25 30 HisTrp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35 40 45 AspThr Phe Lys Leu Ser Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 GlySer Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp 65 70 75 80Asp Phe Ala Thr Tyr Tyr Cys Phe Gln Gly Ser Gly Tyr Pro Phe Thr 85 90 95Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 35 7 PRT Homo sapiens 35Asp Thr Phe Lys Leu Ser Ser 1 5 36 120 PRT Homo sapiens 36 Gln Val ThrLeu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln 1 5 10 15 Thr LeuThr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ala 20 25 30 Gly MetSer Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu 35 40 45 Trp LeuAla Asp Ile Trp Trp Asp Gly Lys Lys Asp Tyr Asn Pro Ser 50 55 60 Leu LysAsp Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val 65 70 75 80 ValLeu Lys Val Thr Asn Met Asp Pro Ala Asp Thr Ala Thr Tyr Tyr 85 90 95 CysAla Arg Asp Met Ile Phe Asn Phe Tyr Phe Asp Val Trp Gly Gln 100 105 110Gly Thr Thr Val Thr Val Ser Ser 115 120 37 16 PRT Homo sapiens 37 AspIle Trp Trp Asp Gly Lys Lys Asp Tyr Asn Pro Ser Leu Lys Asp 1 5 10 15 38106 PRT Homo sapiens 38 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu SerAla Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Ala Ser SerArg Val Gly Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Lys Ala ProLys Leu Leu Ile Tyr 35 40 45 Asp Thr Phe Lys Leu Ser Ser Gly Val Pro SerArg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile SerSer Leu Gln Pro Asp 65 70 75 80 Asp Phe Ala Thr Tyr Tyr Cys Phe Gln GlySer Gly Tyr Pro Phe Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys100 105 39 10 PRT Homo sapiens 39 Ser Ala Ser Ser Arg Val Gly Tyr MetHis 1 5 10 40 120 PRT Homo sapiens 40 Gln Val Thr Leu Arg Glu Ser GlyPro Ala Leu Val Lys Pro Thr Gln 1 5 10 15 Thr Leu Thr Leu Thr Cys ThrPhe Ser Gly Phe Ser Leu Ser Thr Ala 20 25 30 Gly Met Ser Val Gly Trp IleArg Gln Pro Pro Gly Lys Ala Leu Glu 35 40 45 Trp Leu Ala Asp Ile Trp TrpAsp Gly Lys Lys Ser Tyr Asn Pro Ser 50 55 60 Leu Lys Asp Arg Leu Thr IleSer Lys Asp Thr Ser Lys Asn Gln Val 65 70 75 80 Val Leu Lys Val Thr AsnMet Asp Pro Ala Asp Thr Ala Thr Tyr Tyr 85 90 95 Cys Ala Arg Asp Met IlePhe Asn Phe Tyr Phe Asp Val Trp Gly Gln 100 105 110 Gly Thr Thr Val ThrVal Ser Ser 115 120 41 16 PRT Homo sapiens 41 Asp Ile Trp Trp Asp GlyLys Lys Ser Tyr Asn Pro Ser Leu Lys Asp 1 5 10 15 42 106 PRT Homosapiens 42 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser ValGly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Leu Ser Ser Arg Val GlyTyr Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu LeuIle Tyr 35 40 45 Asp Thr Met Tyr Gln Ser Ser Gly Val Pro Ser Arg Phe SerGly Ser 50 55 60 Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu GlnPro Asp 65 70 75 80 Asp Phe Ala Thr Tyr Tyr Cys Phe Gln Gly Ser Gly TyrPro Phe Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 437 PRT Homo sapiens 43 Asp Thr Met Tyr Gln Ser Ser 1 5 44 120 PRT Homosapiens 44 Gln Val Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro ThrGln 1 5 10 15 Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu SerThr Ala 20 25 30 Gly Met Ser Val Gly Trp Ile Arg Gln Pro Pro Gly Lys AlaLeu Glu 35 40 45 Trp Leu Ala Asp Ile Trp Trp Asp Gly Lys Lys Ser Tyr AsnPro Ser 50 55 60 Leu Lys Asp Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys AsnGln Val 65 70 75 80 Val Leu Lys Val Thr Asn Met Asp Pro Ala Asp Thr AlaThr Tyr Tyr 85 90 95 Cys Ala Arg Asp Met Ile Phe Asn Phe Tyr Phe Asp ValTrp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser 115 120 45 16PRT Homo sapiens 45 Asp Ile Trp Trp Asp Asp Lys Lys Ser Tyr Asn Pro SerLeu Lys Asp 1 5 10 15 46 106 PRT Homo sapiens 46 Asp Ile Gln Met Thr GlnSer Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr IleThr Cys Leu Pro Ser Ser Arg Val Gly Tyr Met 20 25 30 His Trp Tyr Gln GlnLys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35 40 45 Asp Thr Met Tyr GlnAla Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr GluPhe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp 65 70 75 80 Asp Phe Ala ThrTyr Tyr Cys Phe Gln Phe Ser Gly Tyr Pro Phe Thr 85 90 95 Phe Gly Gly GlyThr Lys Leu Glu Ile Lys 100 105 47 10 PRT Homo sapiens 47 Leu Pro SerSer Arg Val Gly Tyr Met His 1 5 10 48 120 PRT Homo sapiens 48 Gln ValThr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln 1 5 10 15 ThrLeu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ala 20 25 30 GlyMet Ser Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu 35 40 45 TrpLeu Ala Asp Ile Trp Trp Asp Asp Lys Lys His Tyr Asn Pro Ser 50 55 60 LeuLys Asp Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val 65 70 75 80Val Leu Lys Val Thr Asn Met Asp Pro Ala Asp Thr Ala Thr Tyr Tyr 85 90 95Cys Ala Arg Asp Met Ile Phe Asn Phe Tyr Phe Asp Val Trp Gly Gln 100 105110 Gly Thr Thr Val Thr Val Ser Ser 115 120 49 106 PRT Homo sapiens 49Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 1015 Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Arg Val Gly Tyr Met 20 2530 His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35 4045 Asp Thr Phe Phe Leu Asp Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 5560 Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp 65 7075 80 Asp Phe Ala Thr Tyr Tyr Cys Phe Gln Gly Ser Gly Tyr Pro Phe Thr 8590 95 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 50 7 PRT Homosapiens 50 Asp Thr Phe Phe Leu Asp Ser 1 5 51 120 PRT Homo sapiens 51Gln Val Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln 1 5 1015 Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ala 20 2530 Gly Met Ser Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu 35 4045 Trp Leu Ala Asp Ile Trp Trp Asp Asp Lys Lys Ser Tyr Asn Pro Ser 50 5560 Leu Lys Asp Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val 65 7075 80 Val Leu Lys Val Thr Asn Met Asp Pro Ala Asp Thr Ala Thr Tyr Tyr 8590 95 Cys Ala Arg Asp Met Ile Phe Asn Trp Tyr Phe Asp Val Trp Gly Gln100 105 110 Gly Thr Thr Val Thr Val Ser Ser 115 120 52 106 PRT Homosapiens 52 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser ValGly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Arg Val GlyTyr Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu LeuIle Tyr 35 40 45 Asp Thr Arg Tyr Gln Ser Ser Gly Val Pro Ser Arg Phe SerGly Ser 50 55 60 Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu GlnPro Asp 65 70 75 80 Asp Phe Ala Thr Tyr Tyr Cys Phe Gln Gly Ser Gly TyrPro Phe Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 537 PRT Homo sapiens 53 Asp Thr Arg Tyr Gln Ser Ser 1 5 54 106 PRT Homosapiens misc_feature VL Domain 54 Asp Ile Gln Met Thr Gln Ser Pro SerThr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys SerAla Ser Ser Ser Val Gly Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Pro GlyLys Ala Pro Lys Leu Leu Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser GlyVal Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Glu Phe Thr LeuThr Ile Ser Ser Leu Gln Pro Asp 65 70 75 80 Asp Phe Ala Thr Tyr Tyr CysPhe Gln Gly Ser Gly Tyr Pro Phe Thr 85 90 95 Phe Gly Gly Gly Thr Lys LeuGlu Ile Lys 100 105 55 120 PRT Homo sapiens 55 Gln Val Thr Leu Arg GluSer Gly Pro Ala Leu Val Lys Pro Thr Gln 1 5 10 15 Thr Leu Thr Leu ThrCys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ala 20 25 30 Gly Met Ser Val GlyTrp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu 35 40 45 Trp Leu Ala Asp IleTrp Trp Asp Asp Lys Lys Asp Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg LeuThr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val 65 70 75 80 Val Leu Lys ValThr Asn Met Asp Pro Ala Asp Thr Ala Thr Tyr Tyr 85 90 95 Cys Ala Arg AspMet Ile Phe Asn Trp Tyr Phe Asp Val Trp Gly Ala 100 105 110 Gly Thr ThrVal Thr Val Ser Ser 115 120 56 106 PRT Homo sapiens misc_feature VLDomain 56 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser ValGly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val GlyTyr Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu LeuIle Tyr 35 40 45 Asp Thr Phe Lys Leu Ala Ser Gly Val Pro Ser Arg Phe SerGly Ser 50 55 60 Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu GlnPro Asp 65 70 75 80 Asp Phe Ala Thr Tyr Tyr Cys Phe Gln Gly Ser Gly TyrPro Phe Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 57106 PRT Homo sapiens misc_feature VL Domain 57 Asp Ile Gln Met Thr GlnSer Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr IleThr Cys Ser Ala Ser Ser Ser Val Gly Tyr Met 20 25 30 His Trp Tyr Gln GlnLys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35 40 45 Asp Thr Tyr Lys GlnThr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr GluPhe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp 65 70 75 80 Asp Phe Ala ThrTyr Tyr Cys Phe Gln Gly Ser Gly Tyr Pro Phe Thr 85 90 95 Phe Gly Gly GlyThr Lys Leu Glu Ile Lys 100 105 58 7 PRT Homo sapiens 58 Asp Thr Tyr LysGln Thr Ser 1 5 59 106 PRT Homo sapiens misc_feature VL Domain 59 AspIle Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Gly Tyr Met 20 25 30His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35 40 45Asp Thr Arg Tyr Leu Ser Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp 65 70 7580 Asp Phe Ala Thr Tyr Tyr Cys Phe Gln Gly Ser Gly Tyr Pro Phe Thr 85 9095 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 60 106 PRT Homosapiens 60 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser ValGly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val GlyTyr Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu LeuIle Tyr 35 40 45 Asp Thr Phe Lys Leu Ala Ser Gly Val Pro Ser Arg Phe SerGly Ser 50 55 60 Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu GlnPro Asp 65 70 75 80 Asp Phe Ala Thr Tyr Tyr Cys Phe Gln Gly Ser Phe TyrPro Phe Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 619 PRT Homo sapiens 61 Phe Gln Gly Ser Phe Tyr Pro Phe Thr 1 5 62 106 PRTHomo sapiens misc_feature VL Domain 62 Asp Ile Gln Met Thr Gln Ser ProSer Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr CysSer Ala Ser Ser Ser Val Gly Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys ProGly Lys Ala Pro Lys Leu Leu Ile Tyr 35 40 45 Asp Thr Phe Lys Leu Thr SerGly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Glu Phe ThrLeu Thr Ile Ser Ser Leu Gln Pro Asp 65 70 75 80 Asp Phe Ala Thr Tyr TyrCys Phe Gln Gly Ser Gly Tyr Pro Phe Thr 85 90 95 Phe Gly Gly Gly Thr LysLeu Glu Ile Lys 100 105 63 7 PRT Homo sapiens 63 Asp Thr Phe Lys Leu ThrSer 1 5 64 106 PRT Homo sapiens misc_feature VL Domain 64 Asp Ile GlnMet Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp ArgVal Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Gly Tyr Met 20 25 30 His TrpTyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35 40 45 Asp ThrPhe Arg Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly SerGly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp 65 70 75 80 AspPhe Ala Thr Tyr Tyr Cys Phe Gln Gly Ser Gly Tyr Pro Phe Thr 85 90 95 PheGly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 65 106 PRT Homo sapiens 65Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 1015 Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Gly Tyr Met 20 2530 His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35 4045 Asp Thr Phe Arg Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 5560 Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp 65 7075 80 Asp Phe Ala Thr Tyr Tyr Cys Phe Gln Gly Ser Gly Tyr Pro Phe Thr 8590 95 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 66 7 PRT Homosapiens 66 Asp Thr Phe Arg Leu Ala Ser 1 5 67 120 PRT Homo sapiens 67Gln Val Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln 1 5 1015 Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ala 20 2530 Gly Met Ser Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu 35 4045 Trp Leu Ala Asp Ile Trp Trp Asp Asp Lys Lys His Tyr Asn Pro Ser 50 5560 Leu Lys Asp Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val 65 7075 80 Val Leu Lys Val Thr Asn Met Asp Pro Ala Asp Thr Ala Thr Tyr Tyr 8590 95 Cys Ala Arg Asp Met Ile Phe Asn Trp Tyr Phe Asp Val Trp Gly Ala100 105 110 Gly Thr Thr Val Thr Val Ser Ser 115 120 68 106 PRT Homosapiens 68 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser ValGly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val GlyTyr Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu LeuIle Tyr 35 40 45 Asp Thr Tyr Arg His Ala Ser Gly Val Pro Ser Arg Phe SerGly Ser 50 55 60 Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu GlnPro Asp 65 70 75 80 Asp Phe Ala Thr Tyr Tyr Cys Phe Gln Gly Ser Gly TyrPro Phe Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 697 PRT Homo sapiens 69 Asp Thr Tyr Arg His Ser Ser 1 5 70 106 PRT Homosapiens 70 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser ValGly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val GlyTyr Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu LeuIle Tyr 35 40 45 Asp Thr Tyr Lys Gln Thr Ser Gly Val Pro Ser Arg Phe SerGly Ser 50 55 60 Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu GlnPro Asp 65 70 75 80 Asp Phe Ala Thr Tyr Tyr Cys Phe Gln Gly Ser Gly TyrPro Phe Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 71106 PRT Homo sapiens 71 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu SerAla Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Leu Ser SerSer Val Gly Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Lys Ala ProLys Leu Leu Ile Tyr 35 40 45 Asp Thr Phe Phe His Arg Ser Gly Val Pro SerArg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile SerSer Leu Gln Pro Asp 65 70 75 80 Asp Phe Ala Thr Tyr Tyr Cys Phe Gln GlySer Gly Tyr Pro Phe Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys100 105 72 10 PRT Homo sapiens 72 Ser Leu Ser Ser Ser Val Gly Tyr MetHis 1 5 10 73 7 PRT Homo sapiens 73 Asp Thr Phe Phe His Arg Ser 1 5 74106 PRT Homo sapiens 74 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu SerAla Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Ala Ser SerArg Val Gly Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Lys Ala ProLys Leu Leu Ile Tyr 35 40 45 Asp Thr Leu Leu Leu Asp Ser Gly Val Pro SerArg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile SerSer Leu Gln Pro Asp 65 70 75 80 Asp Phe Ala Thr Tyr Tyr Cys Phe Gln GlySer Gly Tyr Pro Phe Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys100 105 75 7 PRT Homo sapiens 75 Asp Thr Leu Leu Leu Asp Ser 1 5 76 106PRT Homo sapiens 76 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser AlaSer Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser ArgVal Gly Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro LysLeu Leu Ile Tyr 35 40 45 Asp Thr Ser Phe Leu Asp Ser Gly Val Pro Ser ArgPhe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser SerLeu Gln Pro Asp 65 70 75 80 Asp Phe Ala Thr Tyr Tyr Cys Phe Gln Gly SerGly Tyr Pro Phe Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100105 77 7 PRT Homo sapiens 77 Asp Thr Ser Phe Leu Asp Ser 1 5 78 120 PRTHomo sapiens 78 Gln Val Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys ProThr Gln 1 5 10 15 Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser LeuSer Thr Ala 20 25 30 Gly Met Ser Val Gly Trp Ile Arg Gln Pro Pro Gly LysAla Leu Glu 35 40 45 Trp Leu Ala Asp Ile Trp Trp Asp Asp Lys Lys Asp TyrAsn Pro Ser 50 55 60 Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser LysAsn Gln Val 65 70 75 80 Val Leu Lys Val Thr Asn Met Asp Pro Ala Asp ThrAla Thr Tyr Tyr 85 90 95 Cys Ala Arg Asp Met Ile Thr Asn Phe Tyr Phe AspVal Trp Gly Ala 100 105 110 Gly Thr Thr Val Thr Val Ser Ser 115 120 7910 PRT Homo sapiens 79 Asp Met Ile Thr Asn Phe Tyr Phe Asp Val 1 5 10 80109 PRT Homo sapiens 80 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe LeuPhe Pro Pro Lys 1 5 10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr ProGlu Val Thr Cys Val 20 25 30 Val Val Asp Val Ser His Glu Asp Pro Glu ValLys Phe Asn Trp Tyr 35 40 45 Val Asp Gly Val Glu Val His Asn Ala Lys ThrLys Pro Arg Glu Glu 50 55 60 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser ValLeu Thr Val Leu His 65 70 75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr LysCys Lys Val Ser Asn Lys 85 90 95 Ala Leu Pro Ala Pro Ile Glu Lys Thr IleSer Lys Ala 100 105 81 108 PRT Homo sapiens 81 Lys Gly Gln Pro Arg GluPro Gln Val Tyr Thr Leu Pro Pro Ser Arg 1 5 10 15 Glu Glu Met Thr LysAsn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 20 25 30 Phe Tyr Pro Ser AspIle Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 35 40 45 Glu Asn Asn Tyr LysThr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 50 55 60 Phe Phe Leu Tyr SerLys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 65 70 75 80 Gly Asn Val PheSer Cys Ser Val Met His Glu Ala Leu His Asn His 85 90 95 Tyr Thr Gln LysSer Leu Ser Leu Ser Pro Gly Lys 100 105 82 15 PRT Homo sapiens 82 GluPro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 1 5 10 15 83 232PRT Homo sapiens 83 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro ProCys Pro Ala 1 5 10 15 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu PhePro Pro Lys Pro 20 25 30 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu ValThr Cys Val Val 35 40 45 Val Asp Val Ser His Glu Asp Pro Glu Val Lys PheAsn Trp Tyr Val 50 55 60 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys ProArg Glu Glu Gln 65 70 75 80 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val LeuThr Val Leu His Gln 85 90 95 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys LysVal Ser Asn Lys Ala 100 105 110 Leu Pro Ala Pro Ile Glu Lys Thr Ile SerLys Ala Lys Gly Gln Pro 115 120 125 Arg Glu Pro Gln Val Tyr Thr Leu ProPro Ser Arg Glu Glu Met Thr 130 135 140 Lys Asn Gln Val Ser Leu Thr CysLeu Val Lys Gly Phe Tyr Pro Ser 145 150 155 160 Asp Ile Ala Val Glu TrpGlu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 165 170 175 Lys Thr Thr Pro ProVal Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 180 185 190 Ser Lys Leu ThrVal Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 195 200 205 Ser Cys SerVal Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 210 215 220 Ser LeuSer Leu Ser Pro Gly Lys 225 230 84 365 PRT Homo sapiens 84 Met Gly ValPro Arg Pro Gln Pro Trp Ala Leu Gly Leu Leu Leu Phe 1 5 10 15 Leu LeuPro Gly Ser Leu Gly Ala Glu Ser His Leu Ser Leu Leu Tyr 20 25 30 His LeuThr Ala Val Ser Ser Pro Ala Pro Gly Thr Pro Ala Phe Trp 35 40 45 Val SerGly Trp Leu Gly Pro Gln Gln Tyr Leu Ser Tyr Asn Ser Leu 50 55 60 Arg GlyGlu Ala Glu Pro Cys Gly Ala Trp Val Trp Glu Asn Gln Val 65 70 75 80 SerTrp Tyr Trp Glu Lys Glu Thr Thr Asp Leu Arg Ile Lys Glu Lys 85 90 95 LeuPhe Leu Glu Ala Phe Lys Ala Leu Gly Gly Lys Gly Pro Tyr Thr 100 105 110Leu Gln Gly Leu Leu Gly Cys Glu Leu Gly Pro Asp Asn Thr Ser Val 115 120125 Pro Thr Ala Lys Phe Ala Leu Asn Gly Glu Glu Phe Met Asn Phe Asp 130135 140 Leu Lys Gln Gly Thr Trp Gly Gly Asp Trp Pro Glu Ala Leu Ala Ile145 150 155 160 Ser Gln Arg Trp Gln Gln Gln Asp Lys Ala Ala Asn Lys GluLeu Thr 165 170 175 Phe Leu Leu Phe Ser Cys Pro His Arg Leu Arg Glu HisLeu Glu Arg 180 185 190 Gly Arg Gly Asn Leu Glu Trp Lys Glu Pro Pro SerMet Arg Leu Lys 195 200 205 Ala Arg Pro Ser Ser Pro Gly Phe Ser Val LeuThr Cys Ser Ala Phe 210 215 220 Ser Phe Tyr Pro Pro Glu Leu Gln Leu ArgPhe Leu Arg Asn Gly Leu 225 230 235 240 Ala Ala Gly Thr Gly Gln Gly AspPhe Gly Pro Asn Ser Asp Gly Ser 245 250 255 Phe His Ala Ser Ser Ser LeuThr Val Lys Ser Gly Asp Glu His His 260 265 270 Tyr Cys Cys Ile Val GlnHis Ala Gly Leu Ala Gln Pro Leu Arg Val 275 280 285 Glu Leu Glu Ser ProAla Lys Ser Ser Val Leu Val Val Gly Ile Val 290 295 300 Ile Gly Val LeuLeu Leu Thr Ala Ala Ala Val Gly Gly Ala Leu Leu 305 310 315 320 Trp ArgArg Met Arg Ser Gly Leu Pro Ala Pro Trp Ile Ser Leu Arg 325 330 335 GlyAsp Asp Thr Gly Val Leu Leu Pro Thr Pro Gly Glu Ala Gln Asp 340 345 350Ala Asp Leu Lys Asp Val Asn Val Ile Pro Ala Thr Ala 355 360 365 85 365PRT Mus sp. 85 Met Gly Met Pro Leu Pro Trp Ala Leu Ser Leu Leu Leu ValLeu Leu 1 5 10 15 Pro Gln Thr Trp Gly Ser Glu Thr Arg Pro Pro Leu MetTyr His Leu 20 25 30 Thr Ala Val Ser Asn Pro Ser Thr Gly Leu Pro Ser PheTrp Ala Thr 35 40 45 Gly Trp Leu Gly Pro Gln Gln Tyr Leu Thr Tyr Asn SerLeu Arg Gln 50 55 60 Glu Ala Asp Pro Cys Gly Ala Trp Val Trp Glu Asn GlnVal Ser Trp 65 70 75 80 Tyr Trp Glu Lys Glu Thr Thr Asp Leu Lys Ser LysGlu Gln Leu Phe 85 90 95 Leu Glu Ala Leu Lys Thr Leu Glu Lys Ile Leu AsnGly Thr Tyr Thr 100 105 110 Leu Gln Gly Leu Leu Gly Cys Glu Leu Ala SerAsp Asn Ser Ser Val 115 120 125 Pro Thr Ala Val Phe Ala Leu Asn Gly GluGlu Phe Met Lys Phe Asn 130 135 140 Pro Arg Ile Gly Asn Trp Thr Gly GluTrp Pro Glu Thr Glu Ile Val 145 150 155 160 Ala Asn Leu Trp Met Lys GlnPro Asp Ala Ala Arg Lys Glu Ser Glu 165 170 175 Phe Leu Leu Asn Ser CysPro Glu Arg Leu Leu Gly His Leu Glu Arg 180 185 190 Gly Arg Arg Asn LeuGlu Trp Lys Glu Pro Pro Ser Met Arg Leu Lys 195 200 205 Ala Arg Pro GlyAsn Ser Gly Ser Ser Val Leu Thr Cys Ala Ala Phe 210 215 220 Ser Phe TyrPro Pro Glu Leu Lys Phe Arg Phe Leu Arg Asn Gly Leu 225 230 235 240 AlaSer Gly Ser Gly Asn Cys Ser Thr Gly Pro Asn Gly Asp Gly Ser 245 250 255Phe His Ala Trp Ser Leu Leu Glu Val Lys Arg Gly Asp Glu His His 260 265270 Tyr Gln Cys Gln Val Glu His Glu Gly Leu Ala Gln Pro Leu Thr Val 275280 285 Asp Leu Asp Ser Ser Ala Arg Ser Ser Val Pro Val Val Gly Ile Val290 295 300 Leu Gly Leu Leu Leu Val Val Val Ala Ile Ala Gly Gly Val LeuLeu 305 310 315 320 Trp Gly Arg Met Arg Ser Gly Leu Pro Ala Pro Trp LeuSer Leu Ser 325 330 335 Gly Asp Asp Ser Gly Asp Leu Leu Pro Gly Gly AsnLeu Pro Pro Glu 340 345 350 Ala Glu Pro Gln Gly Ala Asn Ala Phe Pro AlaThr Ser 355 360 365 86 7 PRT Homo sapiens 86 Val Leu His Gln Asp Trp Leu1 5 87 6 PRT Homo sapiens 87 Leu Met Ile Ser Arg Thr 1 5 88 9 PRT Homosapiens 88 Met His Glu Ala Leu His Asn His Tyr 1 5 89 5 PRT Homo sapiens89 Gly Gln Pro Glu Asn 1 5 90 6 PRT Homo sapiens 90 Leu Tyr Ile Thr ArgGlu 1 5 91 6 PRT Homo sapiens 91 Leu Tyr Ile Ser Arg Thr 1 5 92 6 PRTHomo sapiens 92 Leu Tyr Ile Ser Arg Ser 1 5 93 6 PRT Homo sapiens 93 LeuTyr Ile Ser Arg Arg 1 5 94 6 PRT Homo sapiens 94 Leu Tyr Ile Ser Arg Gln1 5 95 6 PRT Homo sapiens 95 Leu Trp Ile Ser Arg Thr 1 5 96 6 PRT Homosapiens 96 Leu Tyr Ile Ser Leu Gln 1 5 97 6 PRT Homo sapiens 97 Leu PheIle Ser Arg Asp 1 5 98 6 PRT Homo sapiens 98 Leu Phe Ile Ser Arg Thr 1 599 6 PRT Homo sapiens 99 Leu Phe Ile Ser Arg Arg 1 5 100 6 PRT Homosapiens 100 Leu Phe Ile Thr Gly Ala 1 5 101 6 PRT Homo sapiens 101 LeuSer Ile Ser Arg Glu 1 5 102 6 PRT Homo sapiens 102 Arg Thr Ile Ser IleSer 1 5 103 7 PRT Homo sapiens 103 Thr Pro His Ser Asp Trp Leu 1 5 104 7PRT Homo sapiens 104 Ile Pro His Glu Asp Trp Leu 1 5 105 5 PRT Homosapiens 105 Arg Thr Arg Glu Pro 1 5 106 5 PRT Homo sapiens 106 Asp ProPro Glu Ser 1 5 107 5 PRT Homo sapiens 107 Ser Asp Pro Glu Pro 1 5 108 5PRT Homo sapiens 108 Thr Ser His Glu Asn 1 5 109 5 PRT Homo sapiens 109Ser Lys Ser Glu Asn 1 5 110 5 PRT Homo sapiens 110 His Arg Ser Glu Asn 15 111 5 PRT Homo sapiens 111 Lys Ile Arg Glu Asn 1 5 112 5 PRT Homosapiens 112 Gly Ile Thr Glu Ser 1 5 113 5 PRT Homo sapiens 113 Ser MetAla Glu Pro 1 5 114 9 PRT Homo sapiens 114 Met His Glu Ala Leu Arg TyrHis His 1 5 115 9 PRT Homo sapiens 115 Met His Glu Ala Leu His Phe HisHis 1 5 116 9 PRT Homo sapiens 116 Met His Glu Ala Leu Lys Phe His His 15 117 9 PRT Homo sapiens 117 Met His Glu Ala Leu Ser Tyr His Arg 1 5 1189 PRT Homo sapiens 118 Thr His Glu Ala Leu His Tyr His Thr 1 5

What is claimed is:
 1. A modified IgG comprising an IgG constant domaincomprising one or more amino acid modifications relative to a wild-typeIgG constant domain, wherein the modified IgG has an increased half-lifecompared to the half-life of an IgG having the wild-type IgG constantdomain, and wherein the one or more amino acid modifications are at oneor more of positions 251, 253, 255, 285-290, 308-314, 385-389, and428-435.
 2. A modified non-human IgG comprising a non-human IgG constantdomain comprising one or more amino acid modifications relative to awild-type non-human IgG constant domain, wherein the modified IgG has anincreased half-life compared to the half-life of an IgG having thewild-type non-human IgG constant domain, and wherein the one or moreamino acid modifications are at one or more of positions 251-256,285-290, 308-314, 385-389, and 428-436, with the proviso that the one ormore amino acid modifications do not include substitution with leucineat position 252, serine at position 254, and phenylalanine at position256.
 3. A modified human or humanized IgG comprising a human IgGconstant domain comprising one or more amino acid modifications relativeto a wild-type human IgG constant domain, wherein the modified human orhumanized IgG has an increased half-life compared to the half-life of ahuman or humanized IgG having the wild-type human IgG constant domain,and wherein the one or more amino acid modifications are at one or moreof positions 251-256, 285-290, 308-314, 385-389, and 428-436.
 4. Themodified IgG according to claim 1, 2, or 3, wherein at least one of theamino acid modifications is an amino acid substitution.
 5. The modifiedIgG according to claim 1, 2, or 3, wherein at least one of the aminoacid modifications is an amino acid deletion.
 6. The modified IgGaccording to claim 1, 2, or 3, wherein at least one of the amino acidmodifications is an amino acid insertion.
 7. The modified IgG accordingto claim 1, 2 or 3 which has a higher affinity for FcRn than the IgGhaving the wild-type IgG constant domain.
 8. The modified IgG accordingto claim 7 which has a higher affinity for the FcRn at pH 6.0 than at pH7.4.
 9. The modified IgG according to claim 1, wherein said one or moreamino acid modifications are amino acid substitutions at one or more ofpositions 251, 255, 308, 309, 311, 312, 314, 385, 386, 387, 389, 428,433, 434, or
 436. 10. The modified IgG according to claim 2 or 3,wherein said one or more amino acid modifications are amino acidsubstitutions at one or more of positions 251, 252, 254, 255, 256, 308,309, 311, 312, 314, 385, 386, 387, 389, 428, 433, 434 or
 336. 11. Themodified IgG according to claim 1 or 3 wherein said one or more aminoacid modifications are substitution with leucine at position 251,substitution with tyrosine, tryptophan or phenylalanine at position 252,substitution with threonine or serine at position 254, substitution witharginine at position 255, substitution with glutamine, arginine, serine,threonine, or glutamate at position 256, substitution with threonine atposition 308, substitution with proline at position 309, substitutionwith serine at position 311, substitution with aspartate at position312, substitution with leucine at position 314, substitution witharginine, aspartate or serine at position 385, substitution withthreonine or proline at position 386, substitution with arginine orproline at position 387, substitution with proline, asparagine or serineat position 389, substitution with methionine or threonine at position428, substitution with tyrosine or phenylalanine at position 434,substitution with histidine, arginine, lysine or serine at position 433,or substitution with histidine, tyrosine, arginine or threonine atposition
 436. 12. The modified IgG according to claim 11, wherein saidone or more amino acid substitutions are substitutions with tyrosine atposition 252, threonine at position 254 and glutamate at
 256. 13. Themodified IgG according to claim 11, wherein said one or more amino acidsubstitutions are substitutions with lysine at position 433,phenylalanine at position 434 and histidine at position
 436. 14. Themodified IgG according to claim 11, wherein said amino acid substitutionis a substitution with tyrosine or tryptophan at position
 252. 15. Themodified IgG according to claim 2 wherein said one or more amino acidmodifications are substitution with leucine at position 251,substitution with tyrosine, tryptophan or phenylaline at position 252,substitution with threonine at position 254, substitution with arginineat position 255, substitution with glutamine, arginine, serine,threonine, or glutamate at position 256, substitution with threonine atposition 308, substitution with proline at position 309, substitutionwith serine at position 311, substitution with aspartate at position312, substitution with leucine at position 314, substitution witharginine, aspartate or serine at position 385, substitution withthreonine or proline at position 386, substitution with arginine orproline at position 387, substitution with proline, asparagine or serineat position 389, substitution with methionine or threonine at position428, substitution with tyrosine or phenylalanine at position 434,substitution with histidine, arginine, lysine or serine at position 433,or substitution with histidine, tyrosine, arginine or threonine atposition
 436. 16. The modified IgG according to claim 15, wherein saidone or more amino acid substitutions are substitution with tyrosine atposition 252, threonine at position 254 and glutamate at
 256. 17. Themodified IgG according to claim 15, wherein said one or more amino acidsubstitutions are substitution with lysine at position 433,phenylalanine at position 434 and histidine at position
 436. 18. Themodified IgG according to claim 15, wherein said amino acid substitutionis a substitution with tyrosine or tryptophan at position
 252. 19. Themodified IgG according to claim 3 or 15 which has the heavy chainvariable domain and light chain variable domain of SYNAGIS®.
 20. Themodified IgG according to claim 3 or 15 which has the heavy chainvariable domain and light chain variable domain of AFFF, p12f2, p12f4,p11d4, Ale109, A12a6, A13c4, A17d4, A4B4, A8C7, 1X-493L1FR, H3-3F4,M3H9, Y10H6, DG, AFFF(1), 6H8, L1-7E5, L215B10, A13A11, A1H5, A4B4(1),A4B4L1FR-S28R, or A4B4-F52S.
 21. A fusion protein comprising a non-IgGpolypeptide covalently linked to a modified IgG constant domain, or afragment thereof that binds to FcRn, said modified IgG constant domainor fragment comprising one or more amino acid modifications relative toa wild-type IgG constant domain, wherein the one or more modificationsare at one or more of positions 251, 253, 255, 285-290, 308-314,385-389, and 428-436, and wherein said fusion protein has a longer halflife than the non-IgG polypeptide alone.
 22. A fusion protein comprisinga non-IgG polypeptide covalently linked to a modified non-human IgGconstant domain, or a fragment thereof that binds to FcRn, said modifiednon-human IgG constant domain or fragment comprising one or more aminoacid modifications relative to a wild-type non-human IgG constantdomain, wherein the one or more modifications are at one or more ofpositions 251-256, 285-290, 308-314, 385-389, and 428-436, with theproviso that the modified amino acid sequence does not have leucine atposition 252, serine at position 254, and phenylalanine at position 256,and wherein said fusion protein has a longer half life than the non-IgGpolypeptide alone.
 23. A fusion protein comprising a non-IgG polypeptidecovalently linked to a modified human IgG constant domain, or a fragmentthereof that binds to FcRn, said modified human IgG constant domain orfragment comprising one or more amino acid modifications relative to awild-type human IgG constant domain, wherein the one or moremodifications are at one or more of positions 251-256, 285-290, 308-314,385-389, and 428-436, and wherein said fusion protein has a longer halflife than the non-IgG polypeptide alone.
 24. The fusion proteinaccording to claim 21, 22, or 23, wherein at least one of the amino acidmodifications is an amino acid substitution.
 25. The fusion proteinaccording to claim 21, 22, or 23, wherein at least one of the amino acidmodifications is an amino acid deletion.
 26. The fusion proteinaccording to claim 21, 22, or 23, wherein at least one of the amino acidmodifications is an amino acid insertion.
 27. The fusion proteinaccording to claim 21, 22, or 23, wherein the modified IgG constantdomain or fragment has an increased affinity for FcRn relative to thewild-type IgG constant domain.
 28. The fusion protein according to claim27, wherein the modified IgG constant domain or fragment has a higheraffinity for the FcRn at pH 6.0 than at pH 7.4.
 29. The fusion proteinaccording to claim 21, 22, or 23, wherein the non-IgG polypeptide is animmunoglobulin.
 30. The fusion protein according to claim 21, whereinthe one or more amino acid modifications are amino acid substitutions atone or more of positions 251, 255, 308, 309, 311, 312, 314, 385, 386,387, 389, 428, 433, 434, or
 436. 31. The fusion protein according toclaim 22 or 23, wherein the one or more amino acid modifications areamino acid substitutions at one or more of positions 251, 252, 254, 255,256, 308, 309, 311, 312, 314, 385, 386, 387, 389, 428, 433, 434 or 336.32. The fusion protein according to claim 21 or 23, wherein said one ormore amino acid modifications are substitution with leucine at position251, substitution with tyrosine, tryptophan or phenylalanine at position252, substitution with threonine or serine at position 254, substitutionwith arginine at position 255, substitution with glutamine, arginine,serine, threonine, or glutamate at position 256, substitution withthreonine at position 308, substitution with proline at position 309,substitution with serine at position 311, substitution with aspartate atposition 312, substitution with leucine at position 314, substitutionwith arginine, aspartate or serine at position 385, substitution withthreonine or proline at position 386, substitution with arginine orproline at position 387, substitution with proline, asparagine or serineat position 389, substitution with methionine or threonine at position428, substitution with tyrosine or phenylalanine at position 434,substitution with histidine, arginine, lysine or serine at position 433,or substitution with histidine, tyrosine, arginine or threonine atposition
 436. 33. The fusion protein according to claim 32, wherein saidone or more amino acid substitutions are substitutions with tyrosine atposition 252, threonine at position 254 and glutamate at
 256. 34. Thefusion protein according to claim 32, wherein said one or more aminoacid substitutions are substitutions with lysine at position 433,phenylalanine at position 434 and histidine at position
 436. 35. Thefusion protein according to claim 32, wherein said amino acidsubstitution is a substitution with tyrosine or tryptophan at position252.
 36. The fusion protein according to claim 32, wherein said one ormore amino acid modifications are substitution with leucine at position251, substitution with tyrosine, tryptophan or phenylalanine at position252, substitution with threonine at position 254, substitution witharginine at position 255, substitution with glutamine, arginine, serine,threonine, or glutamate at position 256, substitution with threonine atposition 308, substitution with proline at position 309, substitutionwith serine at position 311, substitution with aspartate at position312, substitution with leucine at position 314, substitution witharginine, aspartate or serine at position 385, substitution withthreonine or proline at position 386, substitution with arginine orproline at position 387, substitution with proline, asparagine or serineat position 389, substitution with methionine or threonine at position428, substitution with tyrosine or phenylalanine at position 434,substitution with histidine, arginine, lysine or serine at position 433,or substitution with histidine, tyrosine, arginine or threonine atposition
 436. 37. The fusion protein according to claim 36, wherein saidone or more amino acid substitutions are substitutions with tyrosine atposition 252, threonine at position 254 and glutamate at
 256. 38. Thefusion protein according to claim 36, wherein said one or more aminoacid substitutions are substitutions with lysine at position 433,phenylalanine at position 434 and histidine at position
 436. 39. Thefusion protein according to claim 36, wherein said amino acidsubstitution is a substitution with tyrosine or tryptophan at position252.
 40. A molecule comprising a non-protein agent conjugated to amodified IgG constant domain, or a fragment thereof that binds to FcRn,said modified IgG constant domain or fragment comprising one or moreamino acid modifications relative to a wild-type IgG constant domain,wherein the one or more modifications are at one or more of positions251, 253, 255, 285-290, 308-314, 385-389, and 428-436, and wherein saidmolecule has a longer half life than the non-protein agent alone.
 41. Amolecule comprising a non-protein agent conjugated to a modifiednon-human IgG constant domain, or a fragment thereof that binds to FcRn,said modified non-human IgG constant domain or fragment comprising oneor more amino acid modifications relative to a wild-type non-human IgGconstant domain, wherein the one or more modifications are at one ormore of positions 251-256, 285-290, 308-314, 385-389, and 428-436, withthe proviso that the modified amino acid sequence does not have leucineat position 252, serine at position 254, and phenylalanine at position256, and wherein said molecule has a longer half life than thenon-protein agent alone.
 42. A molecule comprising a non-protein agentconjugated to a modified human IgG constant domain, or a fragmentthereof that binds to FcRn, said modified human IgG constant domain orfragment comprising one or more amino acid modifications relative to awild-type human IgG constant domain, wherein the one or moremodifications are at one or more of positions 251-256, 285-290, 308-314,385-389, and 428-436, and wherein said molecule has a longer half lifethan the non-protein agent alone.
 43. The molecule according to claim40, 41, or 42 wherein at least one of the amino acid modifications is anamino acid substitution.
 44. The molecule according to claim 40, 41, or42 wherein at least one of the amino acid modifications is an amino aciddeletion.
 45. The molecule according to claim 40, 41 or 42, wherein atleast one of the amino acid modifications is an amino acid insertion.46. The molecule according to claim 40, 41 or 42, wherein the modifiedIgG constant domain or fragment has an increased affinity for FcRnrelative to the wild-type constant domain.
 47. The molecule according toclaim 46, wherein the modified IgG constant domain or fragment has ahigher affinity for the FcRn at pH 6.0 than at pH 7.4.
 48. The moleculeaccording to claim 40, wherein the one or more amino acid modificationsare amino acid substitutions at one or more of positions 251, 255, 308,309, 311, 312, 314, 385, 386, 387, 389, 428, 433, 434, or
 436. 49. Themolecule according to claim 41 or 42, wherein the one or more amino acidmodifications are substitution at one or more of positions 251, 252,254, 255, 256, 308, 309, 311, 312, 314, 385, 386, 387, 389, 428, 433,434 or
 336. 50. The molecule according to claim 40 or 42, wherein saidone or more amino acid modifications are substitution with leucine atposition 251, substitution with tyrosine, tryptophan or phenylalanine atposition 252, substitution with threonine or serine at position 254,substitution with arginine at position 255, substitution with glutamine,arginine, serine, threonine, or glutamate at position 256, substitutionwith threonine at position 308, substitution with proline at position309, substitution with serine at position 311, substitution withaspartate at position 312, substitution with leucine at position 314,substitution with arginine, aspartate or serine at position 385,substitution with threonine or proline at position 386, substitutionwith arginine or proline at position 387, substitution with proline,asparagine or serine at position 389, substitution with methionine orthreonine at position 428, substitution with tyrosine or phenylalanineat position 434, substitution with histidine, arginine, lysine or serineat position 433, or substitution with histidine, tyrosine, arginine orthreonine at position
 436. 51. The molecule according to claim 50,wherein said one or more amino acid substitutions are substitutions withtyrosine at position 252, threonine at position 254 and glutamate at256.
 52. The molecule according to claim 50, wherein said one or moreamino acid substitutions are substitutions with lysine at position 433,phenylalanine at position 434 and histidine at position
 436. 53. Themolecule according to claim 50, wherein said amino acid substitution isa substitution with tyrosine or tryptophan at position
 252. 54. Themolecule according to claim 41, wherein said one or more amino acidmodifications are substitution with leucine at position 251,substitution with tyrosine, tryptophan or phenylalanine at position 252,substitution with threonine at position 254, substitution with arginineat position 255, substitution with glutamine, arginine, serine,threonine, or glutamate at position 256, substitution with threonine atposition 308, substitution with proline at position 309, substitutionwith serine at position 311, substitution with aspartate at position312, substitution with leucine at position 314, substitution witharginine, aspartate or serine at position 385, substitution withthreonine or proline at position 386, substitution with arginine orproline at position 387, substitution with proline, asparagine or serineat position 389, substitution with methionine or threonine at position428, substitution with tyrosine or phenylalanine at position 434,substitution with histidine, arginine, lysine or serine at position 433,or substitution with histidine, tyrosine, arginine or threonine atposition
 436. 55. The molecule according to claim 54, wherein said oneor more amino acid substitutions are substitutions with tyrosine atposition 252, threonine at position 254 and glutamate at
 256. 56. Themolecule according to claim 54, wherein said one or more amino acidsubstitutions are substitutions with lysine at position 433,phenylalanine at position 434 and histidine at position
 436. 57. Themolecule according to claim 54, wherein said amino acid substitution isa substitution with tyrosine or tryptophan at position
 252. 58. Apharmaceutical composition comprising the modified human or humanizedIgG according to claim 3 and a pharmaceutically acceptable carrier. 59.A pharmaceutical composition comprising the fusion protein according toclaim 23 and a pharmaceutically acceptable carrier.
 60. A pharmaceuticalcomposition comprising the molecule according to claim 42 and apharmaceutically acceptable carrier.
 61. A method of treating a diseaseor disorder comprising administering to a patient in need thereof atherapeutically effective amount of the modified human or humanized IgGaccording to claim
 3. 62. The method according to claim 61 whichcomprises passive immunotherapy.
 63. A method of treating a disease ordisorder comprising administering to a patient in need thereof atherapeutically effective amount of the fusion protein according toclaim
 23. 64. A method of treating a disease or disorder comprisingadministering to a patient in need thereof a therapeutically effectiveamount of the molecule according to claim
 42. 65. A nucleic acidcomprising a nucleotide sequence encoding the modified IgG constantdomain according to claim 1, 2 or 3, or an FcRn binding fragmentthereof.
 66. A nucleic acid comprising a nucleotide sequence encodingthe fusion protein according to claim 21, 22, or
 23. 67. A host cellcomprising the nucleic acid according to claim
 65. 68. A host cellcomprising the nucleic acid according to claim
 66. 69. A kit comprisingthe modified human or humanized IgG according to claim
 3. 70. A kitcomprising the fusion protein according to claim
 23. 71. A kitcomprising the molecule according to claim
 42. 72. A method ofpreventing a disease or disorder comprising administering to a subject aprophylactically effective amount of the modified human or humanized IgGaccording to claim
 3. 73. The method according to claim 72 which ispassive immunotherapy.
 74. The method according to claim 72 wherein thedisease is RSV infection.
 75. The method according to claim 74, whereinsaid modified human or humanized IgG is a modified SYNAGIS® antibody.76. The method according to claim 74, wherein said modified human orhumanized IgG is a modified AFFF, p12f2, p12f4, p11d4, Ale109, A12a6,A13c4, A17d4, A4B4, A8C7, 1X-493L1FR, H3-3F4, M3H9, Y10H6, DG, AFFF(1),6H8, L1-7E5, L215B10, A13A11, A1H5, A4B4(1), A4B4L1FR-S28R or A4B4-F52Santibody.
 77. A method of vaccinating a subject comprising administeringto said subject an amount of the modified human or humanized IgGaccording to claim 3 effective to elicit an immune response.
 78. Amethod of vaccinating a subject comprising administering to said subjectan amount of the fusion protein according to claim 23 effective toelicit an immune response.
 79. The method according to 61 whichcomprises administrating a dose of said modified human or humanized IgGthat is lower than the lowest therapeutically effective dose of a secondIgG identical to said modified human or humanized IgG except that saidsecond IgG lacks said one or more amino acid modifications.
 80. Themethod according to claim 79 which results in fewer or less severe sideeffects than administration of the therapeutically effective dose of thesecond IgG.
 81. The method according to claim 61 which comprisesadministering a therapeutically effective dosing schedule having lessfrequent doses of said modified human or humanized IgG than thetherapeutically effective dosing schedule having the least frequentdosing of a second IgG identical to said modified human or humanized IgGexcept that said second IgG lacks said one or more amino acidmodifications.
 82. The method according to claim 72 which comprisesadministrating a prophylactically effective dose of said modified humanor humanized IgG that is lower than the lowest prophylacticallyeffective dose of a second IgG identical to said modified human orhumanized IgG except that said second IgG lacks said one or more aminoacid modifications.
 83. The method according to claim 82 which resultsin fewer or less severe side effects than administration of theprophylactically effective dose of the second IgG.
 84. The methodaccording to claim 72 which comprises administering a prophylacticallyeffective dosing schedule having less frequent doses of said modifiedhuman or humanized IgG than the prophylactically effective dosingschedule with the least frequent dosing of a second IgG identical tosaid humani or humanized IgG except that said second IgG lacks said oneor more amino acid modifications.
 85. A method of in vivo diagnosis in asubject comprising: (a) administering to a subject an effective amountof the modified human or humanized IgG according to claim 3 labeled witha detectable marker, said modified human or humanized IgG specificallybinding to an antigen associated with a disease or disorder; (b)allowing the modified human or humanized IgG to concentrate at sites insaid subject where said antigen is found; and (c) detecting saiddetectable marker, whereby detection of said detectable marker above abackground or standard level indicates that the subject has said diseaseor disorder.
 86. The modified human or humanized IgG according to claim3 which immunospecifically binds to an RSV antigen.